We're researching the cytotoxic potential of a plant extract using the onion root tip assay. We have 4 set-ups; 75%, 50%, 25%, and the negative control (water). We let the onions grow for 48 hours before staining the root tips and viewing it under a microscope.
It's our 3rd attempt, but we barely saw cells undergoing mitosis, not even in the negative control. What are we doing wrong? Is there a specific length to the root tip to see mitotic cells? Is there a specific time to harvest them (we tried 8am to 10am)? Thank you so much in advance!
There are two ways of how to carry out the Allium-test (onion root cells test).
In the first variant you can grow the onions directly in the medium (water) that contain the studied chemical and after a few (3-5) days measure the length of the roots – this is a cytotoxicity test that indicates was your chemical cytotoxic or not. This variant is not very good to study genotoxicity.
The second variant – is good to check a genotoxicity. In this case you should grow the onions without any chemical for two-three days to have near 1-2 cm long roots. And only after that you have to put them to a fresh medium that contains a chemical of interest and after 2-72 of growth (depends on the chemical you use) cut the tips of the roots and prepare them for the microscopy analysis.
Yes, not getting any cell stage is frustrating while preparing onion root tip slides. I have experience that. Your problem is not about the preparation and the kinds of test as described by Serge Voychuk , or seasonal condition by Asha Khanna but about the harvesting time. I don't do cytotoxicity/genotoxicity test, but we produce nice preparations throughout the year. We routinely harvest the the root tip in early morning, soon after sunrise. In my time zone at 0600 h. We immediately fix them and enjoy our breakfast leisurely.
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