OWENS, SHIRLEY A.*, JOANNE H. WHALLON, AND STANLEY L. FLEGLER. Laser Scanning Microscope Lab, and Center for Electron Optics, Michigan State University, East Lansing, MI, 48824. - The formation of calcium crystals during fixation of sectioned pulvinus tissue.
Crystal masses formed on the top and within the cells of sectioned
Cercis canadensis (Fabaceae) pulvinus tissue during preparation
for SEM, were determined to form during the fixation process. The
number of crystals is greater when sections are fixed in fresh FAA
(formaldehyde, acetic acid, ethanol) than in 4% glutaraldehyde in a
sodium phosphate buffer. Normal crystal formation, a single druse
crystal within a vacuole, was observed in samples that were not fixed
or fixed and then sectioned. Analysis of the crystals using
energy-dispersive spectroscopy (EDS) showed peaks for calcium. Time
series experiments in which sectioned pulvinus tissue was fixed in FAA
and in 50% ethanol for 30 minutes up to 24 hours showed that crystal
formation occurred within 30 minutes in both the FAA and in the 50%
ethanol. Crystal development was followed microscopically. A
pulvinus is a differentiated motor organ that is responsible for leaf
movement. Differences in the turgor pressure between regions of
cortical cells within the pulvinus causes it to bend and straighten..
Pressure changes are correlated with the movement of ions such as K+,
Ca2+, Cl-, and malate - across cell membranes. The calcium in the
crystals formed comes from the plant itself. but the other components
could come from either the plant or the fixatives. Calcium chloride
is soluble in alcohol, acetone and acids (all fixatives), calcium
oxalate and calcium malate are soluble in all but acetic acid (FAA and
50% ethanol fixatives) and calcium phosphate (buffer in glutaraldehyde
fixative) is insoluble in alcohol. Other components of the calcium
crystals will be analyzed in the future. Meanwhile, considering the
number of calcium crystals formed, calcium is one of the major ions
involved in the movement of the pulvinus of C. canadensis.
Key words: calcium crystals, Cercis canadensis, EDS, fixation, pulvinus