I am trying to count the chromosomes of Polypodium vulgare using a microscope (10*100X, Olympus BX51T), but I am facing some issues.
I am not sure if all cells are in interphase or if some are in metaphase, but the low resolution of the microscope and the small size of the chromosomes make it difficult to distinguish them.
Could anyone please tell me what the real problem is and suggest possible solutions?
Any advise in this regard would be greatly appreciated.
Material: Polypodium vulgare root tip(2n=74, I think the chromosome size is less than 5 μm)
Method: 1)Metaphase arresting - 0.02 M 8-hydroxyquinoline, 4h
2)fixation - Carnoy's solution, 24h
3)cell dissociation - 1 N HCl, 30s in 60°C
4)staining - aceti-orcein, 10min
You need to better dissociate the tissue and you will need a higher magnification.
Try this protocol and/or get in touch with the authors of the publication.
Article Chromosome numbers of Polish ferns
Also, even in the meristems, only a small percentage of the cells will have condensed chromosomes. Many will be in "interphase" still. I don't know if fern root meristems are comparable to Arabidopsis or onion, but if so you should focus on the "zone of cell division" as opposed to the root cap, zone of elongation, etc. Even then,
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