Microforks for transferring small organisms, organs, or tissues into culture.

Techniques have been developed for making microforks from the eye ends of sewing needles. Details are presented for constructing, sterilizing and manipulating these durable, simply designed transfer tools.

Diffusion and Electric Mobility of Ions within Isolated Cuticles of Citrus aurantium: Steady-State and Equilibrium Values.

We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl(-) salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H(+), Li(+), Na(+), K(+), and Cs(+) ranged from 2 x 10(-8) to 0.6 x 10(-8) meters per second when cuticles were bathed in 2 moles per cubic meter Cl(-) salts. The permeability of Cl(-) was 3 x 10(-9) meters per second. The permeability of Li(+), Na(+), and K(+) was about five times less when measured in 500 moles per cubic meter Cl(-) salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle.

Leaf cuticles behave as asymmetric membranes : evidence from the measurement of diffusion potentials.

Cuticles were isolated enzymatically from the leaves of two maple species (Acer saccharum Marsh and A. platanoides L.) and from orange (Citrus aurantium L.). The cuticles were placed in a plastic cuvette and different concentrations of KCl were perfused over the physiological inner and outer surfaces while the electrical potential (E(10)) that developed across the cuticles and was caused by ion diffusion was measured. E(10) was always positive, indicating that the permeability of K(+) was always greater than that of Cl(-). Measured E(10) in cuticles did not fit the Goldman equation, whereas, E(10) measured during KCl diffusion across selected artificial membranes fit the equation. The magnitude of E(10) in cuticles and artificial membranes also was dependent on ionic strength, decreasing as ionic strength increased. These observations are explained by combining classical transport equations with equations that describe the equilibrium ion distribution between ionic double layers in the cuticle or membranes and the bathing solution.

Movement of Cations through Cuticles of Citrus aurantium and Acer saccharum: Diffusion Potentials in Mixed Salt Solutions.

We examined some biophysical mechanisms of ion migration across leaf cuticles enzymatically isolated from Acer saccharum L. and Citrus aurantium L. leaves. Diffusion potential measurements were used to calculate the permeabilities of Cl(-), Li(+), Na(+), and Cs(+) ions all as a ratio with respect to the permeability of K(+) in cuticles. In 2 millimolar ionic strength solutions the permeability sequence from high to low was K = Cs > Na > Li >> Cl. When the outer and inner surfaces of cuticles were bathed in artificial precipitation and artificial apoplast, respectively, diffusion potentials ranging from -52 to -91 millivolts were measured (inside negative). The Goldman equation predicted that the measured potentials were enough to increase the driving force on the accumulation of heavy metals by a factor of 4 to 7. Other ions migrate with forces 3 to 10 times less than predicted by the Goldman equation for concentration differences alone. Our analysis showed that Ca(2+), and perhaps Mg(2+), might even be accumulated against concentration gradients under some circumstances. Their uptake was apparently driven by the diffusion potentials created by the outward migration of monovalent salts. We feel that future models predicting leaching of nutrients from trees during acid rain events must be modified to account for the probable influence of diffusion potentials on ion migration.

Diffusion and Electric Mobility of KCI within Isolated Cuticles of Citrus aurantium.

Fick's second law has been used to predict the time course of electrical conductance change in isolated cuticles following the rapid change in bathing solution (KCI) from concentration C to 0.1 C. The theoretical time course is dependent on the coefficient of diffusion of KCI in the cuticle and the cuticle thickness. Experimental results, obtained from cuticles isolated from sour orange (Citrus aurantium), fit with a diffusion model of an isolated cuticle in which about 90% of the conductance change following a solution change is due to salts diffusing from polar pores in the wax, and 10% of the change is due to salt diffusion from the wax. Short and long time constants for the washout of KCI were found to be 0.11 and 3.8 hours, respectively. These time constants correspond to KCI diffusion coefficients of 1 x 10(-15) and 3 x 10(-17) square meters per second, respectively. The larger coefficient is close to the diffusion coefficient for water in polar pores of Citrus reported elsewhere (M Becker, G Kerstiens, J Schönherr [1986] Trees 1: 54-60). This supports our interpretation of the washout kinetics of KCI following a change in concentration of bathing solution.