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Questions and Answers
What is the primary purpose of the Feulgen staining technique?
What is the primary purpose of the Feulgen staining technique?
What occurs to DNA during the Feulgen reaction after hydrolysis?
What occurs to DNA during the Feulgen reaction after hydrolysis?
Why does acetic acid and alcohol in a 1:3 ratio act as a fixative for root tips?
Why does acetic acid and alcohol in a 1:3 ratio act as a fixative for root tips?
In which plant tissues is mitotic cell division predominantly found?
In which plant tissues is mitotic cell division predominantly found?
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What color complex is produced when the Feulgen reagent reacts with aldehyde groups from DNA?
What color complex is produced when the Feulgen reagent reacts with aldehyde groups from DNA?
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What is the purpose of washing the roots with distilled water after using HCl?
What is the purpose of washing the roots with distilled water after using HCl?
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What can occur if hydrolysis is excessive during the preparation of the root tips?
What can occur if hydrolysis is excessive during the preparation of the root tips?
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What is indicated by the presence of purple dye after staining the hydrolyzed root tips?
What is indicated by the presence of purple dye after staining the hydrolyzed root tips?
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What temperature is recommended for the water bath step during hydrolysis?
What temperature is recommended for the water bath step during hydrolysis?
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Which stage of mitosis is NOT mentioned in the provided content?
Which stage of mitosis is NOT mentioned in the provided content?
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Study Notes
Feulgen Squash Technique
- A staining technique used in cyto-histology to locate DNA in dividing cells (mitotic cell division).
- Developed by Robert Feulgen.
- DNA is visualized using optical microscopy after acid hydrolysis.
- Mitotic cell division is prominent in meristematic tissue.
- Meristematic tissue is found in shoot apices, and axillary buds in plants, and root tips.
Principle of Feulgen Reaction
- DNA in cells is demonstrated after HCL hydrolysis.
- Aldehyde groups from DNA are released.
- Feulgen reagent reacts with these aldehyde groups, forming a magenta/purple color complex.
- This complex (stained DNA chromatin) is visible under a microscope.
Practical Session
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Stages of Germination & Pre-Treatment:
- Soaking seeds in tap water for 24 hours.
- Placing soaked seeds in irrigated sand or sawdust for 3 days.
- Cutting off root tips (0.5-1cm) from germinated seeds.
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Fixation:
- Transferting collected root tips immediately to fixative solution (acetic acid:alcohol = 1:3).
- Fixing for 24 hours.
- Storing in 70% ethanol in the refrigerator.
- The combination of chemicals preserves the cell structures/components, and stops cell division and growth.
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Hydrolysis:
- Washing root tips with distilled water to remove fixative.
- Placing tips in 1N HCL for a determined period of time in a water bath (at a specific temperature).
- Transfering the hydrolyzed roots to a dry test tube.
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Why Hydrolysis is Important:
- Less Hydrolysis: Releases fewer aldehyde groups, leading to less staining.
- Optimal Hydrolysis: Releases the optimal amount of aldehyde groups, resulting in the most effective staining, producing max purple dye, and optimal outcomes.
- Excess Hydrolysis: Causes structural damage to nucleic acids, decreasing the staining's value/success rate.
Staining and Slide Preparation
- Removing the root tips from the staining solution and placing it on a slide.
- Adding 2ml Feulgen reagent for 30 minutes.
- Squashing the root tip with a pencil eraser and adding 1-2 drops of 45% acetic acid.
- Examining under a microscope.
Results (Under Microscope)
- Images of stained root tips visible under a microscope, showcasing different stages of mitosis.
Stages of Mitosis
- Images (micrographs) showing the various stages of mitosis (Prophase, Metaphase, Anaphase, and Telophase).
- Students should illustrate the stages with drawings and labels.
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Description
Explore the Feulgen squash technique used in cyto-histology to locate DNA in dividing cells. Learn about the principle of the Feulgen reaction and the practical steps involved in preparing samples for microscopic analysis. This quiz delves into the process of visualizing DNA in mitotic cell division, particularly in plant tissues.
