Anyone know the best way to stain Phaseolus vulgaris (common beans) for cytogenetics? I would like to stain meristematic cells from root tips for studying chromosome aberrations and micronuclei.
@C. K. Grisolia,
I think for staining, first you cut the tip of the root and squeeze the semi-liquid material on the glass slide with the help of needle. Then add a drop of 1 or 2% acetocarmine stain. OR you can cut the tip of the root into very fine small pieces and add a drop of 1 or 2% acetocarmine stain. After this heat the slide gently. Now spread the cells by putting a thumb pressure on the cover slip under the folds of the blotting sheet. Observe the slide under microscope for chromosomes.
OR
Give gentle heating to few roots by putting them in a watch glass containing acetocarmine stain. Now the roots will get stained with acetocarmine. Now squeeze the semi-liquid material from the root OR cut the root into small pieces. Add a drop of 45% acetic acid to the material and cover it with cover slip. Follow rest of the procedure same as above.
You cut root tips between 10 to 11 am. Hydrolyse with 5N HCl at room temp. for 30 mins. wash with water. Mix 1:1 acetoorcine and acetocarmine stain in petridish and put root tips for 30 mins. Wash in 45% acetic acid very brief and then make squash after passing gentle on spirit lamp. You will get well spread stages of mitoses .
Roots cut and fixed in ethanol–acetic acid (3:1 v ⁄ v) at 4 C for 24 h, then stained with
acetic orcein, after this heat the slide gently, spread the cells by putting a thumb pressure on the cover slip under the folds of the blotting sheet. Observe the slide under microscope for chromosomes and micronuclei
The procedures I have read are all good. My own contribution is that the initial elongation at germination in many beans is due to turgor pressure. Wait for two days after emergence so you can have a god mitotic index.
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