Lyophilization of cotton leaves

1. Freeze cotton leaves sample at -70 C in a deep freezer. - Transport the sample in an ice chest box. - Check to see that drain plug is closed (left side of condenser) and be sure that switch is off and ballast open. - Turn on bottom switch of the condenser. - Turn on top switches of chamber and set "shelf temperature control" to -400 or -50 C. Then, wait for chamber temperature to drop to set level (it usually takes 1 to 3 hr). - Turn on bottom switch. - Place the frozen samples on trays. Arrange in even layers. Placing second layer of tubes in bottom 2 trays is OK). It may be fun to place the probes into samples to watch the changing temperature. - Close the chamber and wait for 1 to 2 minutes till the samples and chamber temperature equalize. During this time, ice crystals will coat metal parts of the chamber. - Turn on switch and close ballast, and wait for vacuum to reach <100 mT (about 5 to 10 min). In the mean time, condenser temperature should be at least -40 C. - Set "shelf temperature control" to -20 C and let run for 3 hr at least or overnight. - Check the condenser periodically to be sure that the coil has not collected enough ice to lug it up (rarely a problem on a fresh run). - After the run is complete, turn off switch and open ballast. - Turn on and turn off vacuum switches simultaneously. - Remove the samples and turn off top switches. - Turn off switch. - Close the cap of samples tightly immediately. - Place samples in dry, air tight environment as soon as possible and store out of direct sunlight.