3.3.2. Aceto-orcein and Aceto-carmine stain preparation

Aceto-orcein : 2gm of orcein is dissolved in 100 ml hot 45% glacial acetic acid, shake well and filter the stock solution. Mix 9 parts of orcein with 1 part of 1N HCL.

Aceto-carmine : 0.5% carmine is dissolved in 100 ml hot 45% glacial acetic acid, boil for half an hour and finally cool and filter.    

Staining protocol

• 1. On a microscopic slide, the material fixed priorly with the fixative is placed and a drop of aceto-orcein or aceto-carmine is added.

2. Slide is warmed over a spirit lamp keeping in mind that the temperature should not increase beyond 60ºC.

3. Stain is removed with the help of a piece of filter paper, 45%acetic acid is added.

4. Material is covered with the cover slip and squash is prepared.

5. In order to avoid drying of chromosomes the edges of cover slip are sealed with a nail varnish or acid gelatin for temporary vision or keeping slide as a permanent one.

Immature stage of pollen anther and flower bud can be well visualised using Carmine staining method. Heating of the slide during this process causes cells to swell resulting in chromosome separation preventing their overlapping. Orcein works well for staining the microspores in Brassica.

3.3.3. Fluorescent staining with DAPI (4'6-diamino-2 phenylindole)

DAPI solution preparation: 100 μg of DAPI is dissolved in 1ml of distilled water; reagent is stored in an aliquot for years at -20ºC. Working solution can be prepared from stock in McIlvaine buffer to a final concentration of 2-4µl/ml (w/v).

Mcilvaine buffer preparation

Mix (0.1M citric acid (18 ml) with 0.2M Na₂HPO₄ x 2H₂O (82ml) and adjust the at pH at 7.0.

 

Staining protocol

• 1. 50μl of DAPI working solution is added to the slide at room temperature in dark for the duration of about half an hour.

2. Slide is washed with distill water and air dried.

3. Slide is then mount with antifade buffer Citifluor (AFI, Pelco).

4. DAPI stained chromosomes can be destained with methanol-acetic acid (3:1) for 6-12 hrs, rinsed with methanol and air dried.