Olive leaves were
ground in pestle and mortar in liquid nitrogen. This is a critical
step and in our hands the following protocol gives the finest
plant powder. The mortar was placed in a small ice bucket, slightly
bigger than the mortar, and liquid nitrogen was poured outside
and inside the mortar. It is important that while the bucket
containing the mortar may be filled with liquid nitrogen, only
enough liquid nitrogen should be poured into the mortar to cover
the plant tissue and the base of the pestle. The plant tissue was
gently powdered with the pestle until the liquid nitrogen inside
the mortar had nearly evaporated; liquid nitrogen in the ice bucket
containing the mortar was still filled with liquid nitrogen. Just
when the liquid nitrogen inside the mortar had evaporated, the
plant material was strongly ground with the pestle with maximum
pressure and speed. Within a minute, the hard leaf tissue of olives
was pulverized into a fine powder which was then carefully
transferred with a frozen spatula into a frozen storage tube. In
our hands grinding leaf tissue in standing liquid nitrogen did not
produce fine powder.