Quantitative assessment of chemical fixation, including the use of malachite green, for electron microscopy of vacuolate mesophyll cells from leaves of a grass

SUMMARY Various chemical fixation regimes were studied using leaves of the grass Lolium multiflorum Lam. as a test system. To evaluate the quality of fixation for electron microscopy two parameters were chosen; the extent of cell damage to mesophyll cells and damage and change in size of chloroplasts. Reducing the concentration of glutaraldehyde from 3% to 1% resulted in extensive cell damage. The addition of paraformaldehyde to 1% glutaraldehyde did not totally eliminate cell damage and resulted in dilation of thylakoids within the chloroplasts. In tissue fixed and washed in solutions matched to the total osmolality of the primary fixative solution there was little cell damage but the chloroplasts were larger, diameters 4.5times2.9 μm, than when only the buffer vehicle osmolality was maintained during subsequent washings, diameters 3.5times1.6 μm. Malachite green added to the primary fixative solution abolished cell damage regardless of buffer concentration and resulted in the appearance of osmiophilic droplets on the surface of and within the chloroplasts which were not present in the absence of malachite green. It is suggested that malachite green stabilizes cell membranes during primary fixation.