A comparison of three methods for determining the stomatal density of pine needles

Alternative methods were compared for determining the stomatal density of needles from two pine species. Densities estimated from air-dried, whole needles using a binocular dissecting scope were compared to densities estimated from vacuum-dried, intact needles using a scanning electron microscope and expanded peels (or macerated cuticles) using a compound light microscope. Differences among methods were expected from two sources: (1) expansion and shrinkage as a function of water content, and (2) differences in geometry of the measured surface. Estimates from the dissecting scope were similar to those from scanning electron microscopy (t=0.509, n=21, P:=0.62), presumably because both used dried, but otherwise intact whole needles. Light microscopy estimates, however, were lower than dissecting scope estimates (t=-2.307, n=13, P:=0.04). After adjusting for expansion due to hydration and changes in needle geometry, differences disappeared (t=-1.205, n=13, P:=0.25). These results are an important consideration for researchers reconstructing palaeo-atmospheric conditions and assessing plant response to environmental change.