It all depends which plant you are using. If you are wanting a spread plate i.e all the chromosomes countable then you have to catch the right metaphase plate and for a good spread plate give a pre-treatment of Colchicine (good Company) at a concentration range of 0.1 - 0.5% for 3-5 hrs at 16 degree Celcius. This part you have to standardize. After pretreatment fix the root tips in Carnoy's Solution (1:3 ) Alcohol : Acetic Acid for 24 hrs and then store in 70% alcohol for further use. When preparing the slides only take the milky white meristematic region then stain with whichever procedure you are following. Just take care that the hydrolysis process in 1N HCl is well done so that your tip is soft enough for squashing. Squash the tip after putting the coverslip with thumb or tip of unburnt match stick so as to get a single cell layer. Also their are various types of pretreatment. You can check the book on "Chromosome Technique" by Arun Sharma and Archana Sharma. This is a beautiful book on only techniques of chromosome.

Good luck.

For which system?

Here is the protocol I use. http://www.rosebreeders.org/David%20Zlesak--Chromosome%20Counting.pdf

The key for spreading for me is to have very good technique isolating just the meristematic area and removing all other debris from the slide. Then when you squash, it presses and spreads the cells you do have very well. Debris, etc. can get keep one from flattening the cells enough to get a good spread. I have found letting them sit in acid a bit longer doesn't hurt and softens the cell walls too in order to get a flatter and better spread.

Hi Lucy. Come to me and I will teach you how to do it. You do not need to spend on this 6 months, I will show in three weeks. Before you will come, please, read carrefully my papers and then you can write on private - I will explain you everything. Best regards, Tomasz

It all depends which plant you are using. If you are wanting a spread plate i.e all the chromosomes countable then you have to catch the right metaphase plate and for a good spread plate give a pre-treatment of Colchicine (good Company) at a concentration range of 0.1 - 0.5% for 3-5 hrs at 16 degree Celcius. This part you have to standardize. After pretreatment fix the root tips in Carnoy's Solution (1:3 ) Alcohol : Acetic Acid for 24 hrs and then store in 70% alcohol for further use. When preparing the slides only take the milky white meristematic region then stain with whichever procedure you are following. Just take care that the hydrolysis process in 1N HCl is well done so that your tip is soft enough for squashing. Squash the tip after putting the coverslip with thumb or tip of unburnt match stick so as to get a single cell layer. Also their are various types of pretreatment. You can check the book on "Chromosome Technique" by Arun Sharma and Archana Sharma. This is a beautiful book on only techniques of chromosome.

Good luck.

It depends upon the species of plant and the stage of the seedlings. Specifically some plane species are very difficult to get the right stage for squash and chromosome count. Normally it is suggested to have the late part of prophase for chromosome count as the chromosomes at this stage will not be damaged as in metaphase (without membrane) on squash due to pressure. I have worked on Primula species, crotolaria, wheat, Vigna and rice. All have been used the 3:1 acetic alcohol fixtative and 0.5-1% aceto-carmine stain. Based to my experience, we could know the right stage or not on heating and cooling process with drop of aceto-carmine on slide. The root tip will be swollen and looks floopy. I found aceto-carmine quite working than the aceto-orcein and fixative used the same for both staining methods.

Thank you all very much. I will try all thrse very good suggrstions ann am sure to get good results.

Although you have number of good suggestions but you need to focus on the pre-treatment, for that you can treat your roots with nitrous oxide gas for three hours at pressure of 10 p/inc which will increase the mitotic index in your roots and then you can fix the roots in 90% glacial acetic acid for 10 min and finally in 1:3 acetic acid:alcohol. If you will have a number of cells at metaphase it will provide batter chances for at least few good spreads. Further, spreading vary system to system. If you have hard roots, you need to soften them either with cellulase and pectinase treatment or by other hydrlizing agents like 1N Hcl etc. Thereafter stain the roots with acetocarmine, cut the very tip (root cap) of the root and squeeze out the dividing cells (do not cut the root). If you squeeze out the cell suspension without debris you will observe high quality spreads after squishing them in a drop of 45% acetic acid