3.3.4. Non fluorescent Giemsa staining

Stain preparation

Giemsa stain is prepared by adding 4% giemsa in söerensen phosphate buffer (0.05M Na₂HPO₄ and 0.05M KH₂PO₄ )

Staining protocol

• 1.  To the slide with prefixed material Giemsa stain is added and kept for 20-30 mins.

2.  Slide containing stained material is rinsed with distill water and air dried.

3.  Stained chromosomes can be destained with 96% ethanol for 1-2 hrs.

Provides clear visualization of well spread chromosomes preventing overlapping of chromosomes.

 

3.4. Squash preparation

• 1. Small drop of stain is placed on a slide
• .

2. A root-tip is placed using a tweezer on the very slide with stain.

3. Intensely stained meristematic region of root is cut off using a scalpel; tissue is thereafter teased with the needle.

4. Major root region in root cap consisting of epidermal and sub epidermal region layers is removed. This provides flatter squash to be prepared.

5. The drying of slide should be prevented, a cover slip of size which does not allow the stain to fall is used to cover the material.

6. Slide is gently heated over alcohol flame.

7. The excess of stain, if any, can be gently removed by pressing. Flatten the cells by applying pressure keeping in mind that majority of the cells are in same plane of focus.