Orientation Of Cellulose Fibrils Research Articles

In situ infrared imaging of the local orientation of cellulose fibrils in plant secondary cell walls.

The mechanical and chemical properties of plant cell walls greatly rely on the supramolecular assembly of cellulose fibrils. To study the local orientation of cellulose in secondary plant cell walls, diffraction limited infrared (IR) micro-spectroscopic mapping experiments were conducted at different orientation of transverse leaf section of the grass Sorghum bicolor with respect to the polarization direction of the IR radiation. Two-dimensional maps, based on polarization-sensitive absorption bands of cellulose were obtained for different polarization angles. They reveal a significant degree of anisotropy of the cellulose macromolecules as well as of other biopolymers in sclerenchyma and xylem regions of the cross section. Quantification of the signals assigned to polarization sensitive vibrational modes allowed to determine the preferential orientation of the sub-micron cellulose fibrils in single cell walls. A sample of crystalline nano-cellulose comprising both a single microcrystal as well as unordered layers of nanocrystals was used for validation of the approach. The results demonstrate that diffraction limited IR micro-spectroscopy can be used to study hierarchically structured materials with complex anisotropic behavior.

Mechanical model for the deformation of the wood cell wall

Abstract The molecular mechanisms responsible for the deformation of wood, as well as the mechanical interaction of cell-wall components, such as cellulose, lignin and hemicelluloses, are not well understood. In a recently published experiment [1], we have shown that wood foils and single cells of compression wood of spruce (Picea abies [L.] Karst.) could deform permanently under tensile load via a stick and slip mechanism at the molecular level occurring during shear of the matrix between cellulose microfibrils. The shear originates from the fact that microfibrils are spiralling around the central lumen of the wood cell which can be considered as a hollow tube. During stretching, the microfibril angle between the cell axis and the direction of the cellulose was found to decrease in synchrotron X-ray diffraction experiments, giving rise to shear deformation of the matrix and to a recovery mechanism after irreversible deformation. The corresponding stick and slip mechanism is treated here in the framework of a simple mechanical model which shows that the cellulose fibril orientation might define slip planes for shear deformation, in analogy to the slip planes in single crystals as described by Schmid’s law. Moreover, the effect of fibril reorientation upon straining as well as the possibility of cell torsion are considered in the model and discussed with respect to experimental data.

The impact of alterations in lignin deposition on cellulose organization of the plant cell wall.

BackgroundCoordination of synthesis and assembly of the polymeric components of cell walls is essential for plant growth and development. Given the degree of co-mingling and cross-linking among cell wall components, cellulose organization must be dependent on the organization of other polymers such as lignin. Here we seek to identify aspects of that codependency by studying the structural organization of cellulose fibrils in stems from Arabidopsis plants harboring mutations in genes encoding enzymes involved in lignin biosynthesis. Plants containing high levels of G-lignin, S-lignin, H-lignin, aldehyde-rich lignin, and ferulic acid-containing lignin, along with plants with very low lignin content were grown and harvested and longitudinal sections of stem were prepared and dried. Scanning X-ray microdiffraction was carried out using a 5-micron beam that moved across the sections in 5-micron steps and complete diffraction patterns were collected at each raster point. Approximately, 16,000 diffraction patterns were analyzed to determine cellulose fibril orientation and order within the tissues making up the stems.ResultsSeveral mutations—most notably those exhibiting (1) down-regulation of cinnamoyl CoA reductase which leads to cell walls deficient in lignin and (2) defect of cinnamic acid 4-hydroxylase which greatly reduces lignin content—exhibited significant decrease in the proportion of oriented cellulose fibrils in the cell wall. Distinctions between tissues were maintained in all variants and even in plants exhibiting dramatic changes in cellulosic order the trends between tissues (where apparent) were generally maintained. The resilience of cellulose to degradative processes was investigated by carrying out the same analysis on samples stored in water for 30 days prior to data collection. This treatment led to significant loss of cellulosic order in plants rich in aldehyde or H-lignin, less change in wild type, and essentially no change in samples with high levels of G- or S-lignin.ConclusionsThese studies demonstrate that changes in lignin biosynthesis lead to significant disruption in the orientation and order of cellulose fibrils in all tissues of the stem. These dramatic phenotypic changes, in mutants with lignin rich in aldehyde or H-units, correlate with the impact the mutations have on the enzymatic degradation of the plant cell wall.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0540-z) contains supplementary material, which is available to authorized users.

Tunable Photothermal Actuators Based on a Pre-programmed Aligned Nanostructure.

For various applications, it is challenging but essential to obtain complex tunable mechanical actuations in response to environmental stimuli. Here, a general and effective strategy is developed to produce multiple types of photomechanical actuation (from phototropic/apheliotropic bending to three-dimensional helical buckling) by manipulating the orientation of one-dimensional nanomaterials. These materials are manipulated to mimic plants that generate diverse mechanical motions through the orientation of cellulose fibrils. The photomechanical actuations can be completed in milliseconds and can be performed reversibly without detectable fatigue after 100 000 cycles. This capacity to produce multiple types of photomechanical actuation is further developed to produce complex integrated movements, as demonstrated by a light-manipulated robotic arm and a solar energy harvesting system.

Development of a green binder system for paper products

BackgroundIt is important for industries to find green chemistries for manufacturing their products that have utility, are cost-effective and that protect the environment. The paper industry is no exception. Renewable resources derived from plant components could be an excellent substitute for the chemicals that are currently used as paper binders. Air laid pressed paper products that are typically used in wet wipes must be bound together so they can resist mechanical tearing during storage and use. The binders must be strong but cost-effective. Although chemical binders are approved by the Environmental Protection Agency, the public is demanding products with lower carbon footprints and that are derived from renewable sources.ResultsIn this project, carbohydrates, proteins and phenolic compounds were applied to air laid, pressed paper products in order to identify potential renewable green binders that are as strong as the current commercial binders, while being organic and renewable. Each potential green binder was applied to several filter paper strips and tested for strength in the direction perpendicular to the cellulose fibril orientation. Out of the twenty binders surveyed, soy protein, gelatin, zein protein, pectin and Salix lignin provided comparable strength results to a currently employed chemical binder.ConclusionsThese organic and renewable binders can be purchased in large quantities at low cost, require minimal reaction time and do not form viscous solutions that would clog sprayers, characteristics that make them attractive to the non-woven paper industry. As with any new process, a large-scale trial must be conducted along with an economic analysis of the procedure. However, because multiple examples of “green” binders were found that showed strong cross-linking activity, a candidate for commercial application will likely be found.

Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells

BackgroundIn plants, a complex cell wall protects cells and defines their shape. Cellulose fibrils form a multilayered network inside the cell-wall matrix that plays a direct role in controlling cell expansion. Resolving the structure of this network will allow us to comprehend the relationship of cellulose fibril orientation and growth.The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal microscopy of some 200 nm in xy and 550 nm in z for green light, restricts the direct visualization of cellulose to relatively large bundles, whereas the structure of cellulose microfibrils with their diameter below 10 nm remains unresolved. Over the last decade, several so-called super-resolution microscopy approaches have been developed; in this paper we explore the potential of such approaches for the direct visualization of cellulose.ResultsTo ensure optimal imaging we determined the spectral properties of PFS-stained tissue. PFS was found not to affect cell viability in the onion bulb scale epidermis. We present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata.ConclusionsSuper-resolution light microscopy of PFS-stained cellulose fibrils is possible and the increased resolution over conventional approaches makes it a valuable tool for the investigation of the cell-wall structure. This is one step in method developments that will close the gap to more invasive techniques, such as atomic force and electron microscopy.

Probing the chemical and surface chemical modification of vessel cell walls during bleaching of eucalyptus pulp

Abstract Eucalyptus pulp is increasingly used for the manufacture of printing papers. However, its major drawback, the vessel picking, is still waiting for a solution. The detailed features and the characteristic behaviour of vessel elements (VEs) in pulp and paper processes are poorly understood. This study focusses on the chemistry, surface chemistry and morphology of eucalyptus VEs. These properties were followed through the changes introduced by different stages of elemental chlorine-free (ECF) bleaching, by studying separated VEs. Microprobe X-ray photoelectron spectroscopy (μ-XPS) and field emission-scanning electron microscopy (FE-SEM) were applied to elucidate the surface chemical composition and morphological ultrastructure, respectively. The bulk chemical composition was investigated among others by Py-GC/MS. Lignin was detected in vessels still after completed bleaching sequence, whereas the fibres were lignin-free. The vessel lignin was mainly composed by syringyl-type units. Surface coverage by lignin and amount of surface anionic groups were practically unaffected by bleaching. The vessel cell wall structure was observed to be layered in a complex way with no particular orientation of cellulose fibrils, and the different layers seemed to be exfoliated during different bleaching stages.

Effective Young’s Modulus of Bacterial and Microfibrillated Cellulose Fibrils in Fibrous Networks

The deformation micromechanics of bacterial cellulose (BC) and microfibrillated cellulose (MFC) networks have been investigated using Raman spectroscopy. The Raman spectra of both BC and MFC networks exhibit a band initially located at ≈ 1095 cm(-1). We have used the intensity of this band as a function of rotation angle of the specimens to study the cellulose fibril orientation in BC and MFC networks. We have also used the change in this peak's wavenumber position with applied tensile deformation to probe the stress-transfer behavior of these cellulosic materials. The intensity of this Raman band did not change significantly with rotation angle, indicating an in-plane 2D network of fibrils with uniform random orientation; conversely, a highly oriented flax fiber exhibited a marked change in intensity with rotation angle. Experimental data and theoretical analysis shows that the Raman band shift rate arising from deformation of networks under tension is dependent on the angles between the axis of fibrils, the strain axis, the incident laser polarization direction, and the back scattered polarization configurations. From this analysis, the effective moduli of single fibrils of BC and MFC in the networks were estimated to be in the ranges of 79-88 and 29-36 GPa, respectively. It is shown also that for the model to fit the data it is necessary to use a negative Poisson's ratio for MFC networks and BC networks. Discussion of this in-plane "auxetic" behavior is given.

Mechanical model for fiber-laden membranes

An integrated mechanical model for fiber-laden membranes is presented and representative predictions of relevance to cellulose ordering and orientation in the plant cell wall are presented. The model describes nematic liquid crystalline self-assembly of rigid fibers on an arbitrarily curved fluid membrane. The mechanics of the fluid membrane is described by the Helfrich bending-torsion model, the fiber self-assembly is described by the 2D Landau-de Gennes quadrupolar Q-tensor order parameter model, and the fiber-membrane interactions (inspired by an extension of the 2D Maier-Saupe model to curved surfaces) include competing curvo-philic (curvature-seeking) and curvo-phobic (curvature-avoiding) effects. Analysis of the free energy reveals three fiber orientation regimes: (a) along the major curvature, (b) along the minor curvature, (c) away from the principal curvatures, according to the competing curvo-philic and curvo-phobic interactions. The derived shape equation (normal stress balance) now includes curvature-nematic ordering contributions, with both bending and torsion renormalizations. Integration of the shape and nematic order equations gives a complete model whose solution describes the coupled membrane shape/fiber order state. Applications to cylindrical membranes, relevant to the plant cell wall, shows how growth decreases the fiber order parameter and moves the fibers’ director from the axial direction towards the azimuthal orientation, eventually leading to a state of stress predicted by pure membranes. The ubiquitous 54.7° cellulose fibril orientation with respect to the long axis in a cylindrical plant cell wall is shown to be predicted by the preset model when the ratio of curvo-phobic and curvo-philic interactions is in the range of the cylinder radius.

Plants control the properties and actuation of their organs through the orientation of cellulose fibrils in their cell walls

Plants use the orientation of cellulose microfibrils to create cell walls with anisotropic properties related to specific functions. This enables organisms to control the shape and size of cells during growth, to adjust the mechanical performance of tissues, and to perform bending movements of organs. We review the key function of cellulose orientation in defining structural-functional relationships in cell walls from a biomechanics perspective, and illustrate this by examples mainly from our own work. First, primary cell-wall expansion largely depends on the organization of cellulose microfibrils in newly deposited tissue and model calculations allow an estimate of how their passive re-orientation may influence the growth of cells. Moreover, mechanical properties of secondary cell walls depend to a large extent on the orientation of cellulose fibrils and we discuss strategies whereby plants utilize this interrelationship for adaptation. Lastly, we address the question of how plants regulate complex organ movements by designing appropriate supramolecular architectures at the level of the cell wall. Several examples, from trees to grasses, show that the cellulose architecture in the cell wall may be used to direct the swelling or shrinking of cell walls and thereby generate internal growth stress or movement of organs.

Actuation systems in plants as prototypes for bioinspired devices

Plants have evolved a multitude of mechanisms to actuate organ movement. The osmotic influx and efflux of water in living cells can cause a rapid movement of organs in a predetermined direction. Even dead tissue can be actuated by a swelling or drying of the plant cell walls. The deformation of the organ is controlled at different levels of tissue hierarchy by geometrical constraints at the micrometre level (e.g. cell shape and size) and cell wall polymer composition at the nanoscale (e.g. cellulose fibril orientation). This paper reviews different mechanisms of organ movement in plants and highlights recent research in the field. Particular attention is paid to systems that are activated without any metabolism. The design principles of such systems may be particularly useful for a biomimetic translation into active technical composites and moving devices.

Tensile and compressive stresses in tracheids are induced by swelling based on geometrical constraints of the wood cell

Plants are able to pre-stress their tissues in order to actuate their organs. Here, we demonstrate with two tissue types of the secondary xylem of conifers (normal wood and compression wood of spruce (Picea abies)) that either tensile or compressive stresses can develop in the longitudinal direction during the swelling of the cell wall. This dramatic difference appears to be due mostly to differences in cell geometry and cellulose fibril orientation. A mechanical model was developed to demonstrate swelling experiments with the help of sodium iodide experiments. The reversal of longitudinal extension can be predicted, based on the orientation of the (nearly inextensible) cellulose fibrils and the shape of the cell.

Studies on Convolution of Cotton Fiber [I]-Optical Rotation Analysis of Cotton Fibers Cultivated in Various Countries-

Cotton fiber has its own complicated convolutions caused by helicoidal orientation of cellulose fibrils in the secondary cell membrane. This helicoidal orientation has been deduced to be caused by the earth's rotation during fibril development. To confirm this, the optical rotation power of cotton fibers cultivated in various countries was measured using a polarizing microscope. The optical rotation power of Z-type convolution parts in cotton fibers cultivated in the northern hemisphere was greater than that of the S-type convolution parts. On the other hand, for cotton fibers cultivated in the southern hemisphere, the optical rotation power of the S-type convolution parts was greater than that of the Z-type parts. This tendency was found to differ markedly for cotton fibers cultivated in areas located at different distances from the equator.

Cellulose orientation at the surface of the Arabidopsis seedling. Implications for the biomechanics in plant development

In diffuse growing cells the orientation of cellulose fibrils determines mechanical anisotropy in the cell wall and hence also the direction of plant and organ growth. This paper reports on the mean or net orientation of cellulose fibrils in the outer epidermal wall of the whole Arabidopsis plant. This outer epidermal wall is considered as the growth-limiting boundary between plant and environment. In the root a net transverse orientation of the cellulose fibrils occurs in the elongation zone, while net random and longitudinal orientations are found in subsequent older parts of the differentiation zone. The position and the size of the transverse zone is related with root growth rate. In the shoot the net orientation of cellulose fibrils is transverse in the elongating apical part of the hypocotyl, and longitudinal in the fully elongated basal part. Leaf primordia and very young leaves have a transverse orientation. Throughout further development the leaf epidermis builds a very complex pattern of cells with a random orientation and cells with a transverse or a longitudinal orientation of the cellulose fibrils. The patterns of net cellulose orientation correlate well with the cylindrical growth of roots and shoots and with the typical planar growth of the leaf blade. On both the shoot and the root surface very specific patterns of cellulose orientation occur at sites of specific cell differentiation: trichome–socket cells complexes on the shoot and root hairs on the root.

The cytoskeleton, elongation and the control of elongation.

The epidermis is often considered to be the growthlimiting cell layer in young, growing herbaceous plants (Niklas, 1992). In his classic work, Green (1980) elaborated this idea further for organ development, and attributed a crucial role to the orientation of cellulose fibrils as indicator of mechanical properties. A factual proof for a relation between the mean cellulose orientation and mechanical anisotropy was reported recently (Kerstens et al., 2001). Cortical microtubules are supposed to guide the cellulose synthesising complexes in the plane of the plasma membrane (Giddings and Staehelin, 1991). Changes in the direction of cell expansion are accompanied by changes in the orientation of the cortical microtubules. A role for microtubule reorientation is also expected during changes in the rate of elongation: microtubules then will guide nascent cellulose microfibrils deposition in the longitudinal direction, which will hamper elongation and facilitate cell expansion in another direction (Taiz and Zeiger, 1998). However, this generally accepted relation between microtubules and microfibrils has also been questioned (e.g. Baskin, 2000; Emons and Mulder, 2000; Sugimoto et al., 2000; Williamson, 1990). In the elongation zone of Arabidopsis roots, microtubules in epidermal cells are transverse to the root axis. Towards the end of elongation, when cells enter the root hair initiation zone, microtubules switch to a slightly oblique orientation in atrichoblasts and to very oblique or longitudinal orientations in trichoblasts. The rate of elongation can be affected by different means. When roots are exposed to ethylene or ACC, the precursor of ethylene, the elongation rate is quickly and severely reduced, in a dose-dependent way (Le et al., 2001). This is the result of cells stopping elongation at a much reduced size. As soon as elongation of a cell ceases, microtubules change their orientation from transverse to longitudinal. This rearrangement is cell position bound. In the early part of the elongation zone, cell elongation up to a length of 30 μm is not affected and microtubules keep predominantly to a transverse orientation. Osmotic stress induces similar effects on elongation and microtubule reorientation. Experiments with microtubule drugs, demonstrate that microtubules are essential for elongation to go on but reorientation of microtubules is not involved and only concomitant with the fast control of elongation rate. We propose that the switch in microtubule orientation is a mechanism used by the cells to consolidate the shorter cell size and to inhibit further elongation. In the Arabidopsis hypocotyl, elongation starts at the base and proceeds in an acropetal direction. In darkness, elongation proceeds at a much higher rate than in the light. In the lower half of the hypocotyls, cells grow to about 2.5 times the size they reach in the light, while the cells at the top of the hypocotyl become 6–7 times longer. This is accompanied by striking differences in microtubule levels. In darkness, microtubules are quite immobile and neatly transverse in elongating cells. In the light, however, they are extremely mobile and transverse but also a variety of other microtubule orientations were found. However, in all plants studied, cells that have stopped elongation have fewer microtubules and these generally have a longitudinal orientation. Therefore, we propose that when the elongation rate is maximal (darkness) the wall synthesis activity at the cell membrane recruits all available microtubules in transverse arrays. When the rate of cell elongation is much lower (light) only a fraction of the microtubules is recruited and the remainder of the microtubules can freely change orientation. This leads to the variety of orientations seen in the light. When cells have no longer the possibility to elongate (aged cell at the base) the * Corresponding author. Tel.: +32-3-820-2277; fax: +32-3-820-2271. Cell Biology International 27 (2003) 287–289 Cell Biology International

Cellulose orientation in the outer epidermal wall of angiosperm roots: implications for biosystematics.

The net orientation of cellulose fibrils in the outer epidermal wall of the root elongation zone of 57 angiosperm species belonging to 29 families was determined by means of Congo Red fluorescence and polarization confocal microscopy. The angiosperms can be divided in three groups. In all but four plant families, the net orientation of the cellulose fibrils is transverse to the root axis. Three families, the Poaceae, Juncaceae and Cyperaceae, have a totally different organization. In the root elongation zone of these plants, the net orientation of cellulose fibrils in the outer epidermal wall is parallel with the root axis. In roots of one family, the Arecaceae, an elongation zone in the literal sense of the word is absent and cellulose fibrils are randomly oriented.

Cell Walls at the Plant Surface Behave Mechanically Like Fiber-Reinforced Composite Materials

Using extensiometry and polarization confocal microscopy, we provide here the first confirming evidence that the mean orientation of cellulose fibrils in the composite wall determines its mechanical properties. When there is a preferred orientation of the cellulose fibrils, the cell wall is

Polarization confocal microscopy and congo red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants.

The mean or net preferential orientation of cellulose fibrils in plant cell walls is detected with polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo Red. Single cells, arrays of cells in a tissue, or the epidermis of whole organs can be assayed in vivo. Aerial parts require an extra pectinase treatment because of the cuticle, which is impermeable to aqueous solutions. Peeling off the epidermis can be an elegant alternative, especially for leaves. With this method the net preferential fibril orientation can be related to the symmetry axis of the cell in quantitative terms. Data issuing from this approach are useful in current research on plant biomechanics.

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM

The transfer of the glucosyl moiety from uridine diphosphate glucose in the presence of Acetobacter xylinum cell-free extracts led to the formation of a mixture of alkali-soluble and -insoluble cellodextrins. Typical cellulose fibrils could not be detected by electron microscopy in this product. Immediately after release into the medium, cellulose formed by whole cells is in a "prefibrous" form which passes through Millipore filters of 0.45 and 0.8 micro pore diameter. Non-filtrable fibrils arise from this material probably by a process of crystallization involving no extracellular enzymes. Fibrils formed in shaken cell suspensions intertwine and form aggregates visible to the naked eye. In quiet suspensions pellicles are formed which float on the surface. Soluble Na-carboxymethylcellulose (CMC) is incorporated into cellulose fibrils formed in its presence, probably by a process of co-crystallization. Aggregation of fibrils containing CMC is delayed because of electrostatic repulsion between carboxylic groups. The aggregation time depends on the amount of CMC incorporated, its degree of substitution, the pH of the medium, and the ionic strength. The amount of CMC incorporated depends on the relative concentration CMC/cellulose and on the similarity of the CMC and the cellulose molecules i.e. in molecular weight and the number of carboxyl substitutions. Cellulose pellicles formed in the presence of CMC by unshaken cell suspensions consist of crossed, superimposed layers of parallel oriented cellulose fibrils. The same phenomenon is observed when phosphomannan, but not levan, is substituted for CMC. The biogenesis of oriented cellulose fibrils is envisaged as a process comprising the following steps: polymerization of the monomeric precursor, diffusion of the molecule to crystallization sites, crystallization, and orientation. It is proposed that charged polysaccharides play a role similar to that of CMC in affecting the orientation of cellulose fibrils in the plant cell wall.

ELECTRON MICROSCOPY OF PRIMARY CELL WALLS TREATED WITH PECTIC ENZYMES

THE ELECTRON MICROSCOPE has recently been uced to investigate the fine structure of the cell walls of higher plants (Frey-Wyssling et al., 1948; FreyWyssling, 1951; Miihlethaler, 1949, 1950). These workers have paid particular attention to the distribution and orientation of cellulose fibrils within the walls of meristematic and mature cells, and their results have thrown much light on the subject. Preparation of their material has in some cases involved maceration of tissues by a Waring blendor and extraction of non-cellulosic substances by chemical treatment at high temperatures. Such preparations showed clearly the fibrillar structure of the cellulose component of the cell wall. With some plant material at least, we have found that the fibrillar structure of cellulose may be demonstrated by using solutions of pectic enzymes to remove pectic substances prior to electron microscopy. These enzymes are obtained by culturing an isolate of Bacterium aroideae (Townsend) Stapp in a synthetic medium for 4 days at 25?C. (Wood, 1951). The composition of this medium is as follows: glucose, I per cent; asparagine, 0.25 per cent; KH2PO4, 0.1 per cent; MgSO4. 7H20, 0.03 per cent; CaCO3, 1 per cent. After removal of bacteria and other insoluble material by centrifugation, the remaining solution may be used to macerate a wide variety of plant tissues. The pH of the solution is within the range 7.5-7.8 and sufficiently close to the optimum (8-8.5) so that the solution may be used directly. Although unnecessary for the particular ends in view, partial purification of the macerating enzymes may readily be accomplished by fractional precipitation with acetone, alcohol, or ammonium sulphate. Solutions retain their activity for at least 3 months if kept frozen at -50C. Parenchyma tissues, such as those of turnip root, potato tuber, cucumber fruit, and leaves, are cut into pieces approximately one sq. cm. and not exceeding one mm. thick in the case of bulky tissue. These pieces are then immersed in the macerating solution and infiltrated by placing under a water pump vacuum for a few minutes. After 15-30 min. at room temperature, tissues break down readily on shaking to give a suspension consisting for the most part of individual intact cells. The few remaining groups of cells are readily broken up after further incubation and shaking. The suspension is then washed by repeated centrifugation to remove solutes and after suitable dilution placed on collodion-covered grids, shadowed witb palladium and