Paraffin Method for Root-Tip Chromosome Counts
L. P. Randolph
The reagents employed and the sequence of transfers from fixation to paraffin-ribbon mounts are as follows:
- Fix roots 12 to 24 hours in "Craf" (Chromo-acetic-formalin):
Transfer roots directly from Craf to 75% alcohol, changing several times at half-hour intervals to remove most of the fixing fluid; then to 85% alcohol.
From 85% alcohol to normal butyl alcohol as follows:
- Solution A, Chromic 1 gr., Acetic 7 cc., Water 92 cc.
- Solution B, Formalin 30 cc., Water 70 cc.
- Mix equal parts A and B just before using.
- This fluid was developed primarily for making chromosome counts in the root-tips of maize, but it has proved to be very useful for similar studies in many other plants.
- H20 15 cc., 95% ethyl 50 cc., butyl 35 cc.
- H20 5 cc., 95% ethyl 45 cc., butyl 55 cc.
- Absolute ethyl 25 cc., butyl 75 cc.
- Normal butyl, 3 or 4 changes.
Leave roots at least an hour in each solution, 2-3 hours in pure butyl.
Infiltrate gradually with paraffin: Add melted paraffin (melting point 54-55° C.) in an amount equal to about one-third the volume of the butyl alcohol covering the roots. Add the paraffin slowly so it will solidify on top of the butyl alcohol. Place the receptacle (preferably a 30 or 50 cc. pyrex beaker) containing the roots and butyl-paraffin mixture in a paraffin oven at 56° C. Leave over night. As the paraffin melts it passes slowly to the bottom of the beaker and gradually infiltrates the roots. The next day pour off the butyl-paraffin mixture and add pure liquid paraffin. Repeat 3 or 4 times at hourly intervals.
Embed, cooling the paraffin rapidly in ice water.
Prepare cross-sections 10 to 15 microns in thickness. Spread ribbons on slides and dry for several hours at about 40° C.