Observations on the ultrastructure of the thickened sieve cell wall inPinus strobus L.

Summary The thickened sieve cell wall of white pine is shown to comprise a crossed-helical polylamellate structure in which the predominant microfibrillar orientation is greater than 45° with respect to the cell axis. The previously reported observation that microfibrils may be oriented other than parallel to the plane of the cell wall is disputed and it is demonstrated that such an appearance may derive from appropriately oblique sectioning of the wall.     

成熟玉米秆组织结构切片方法的比较研究

于玉米成熟期选择健壮茎秆的基部第三节间为材料,采用徒手切片法、冰冻切片法、石蜡切片法、薄切片法等4种方法,比较不同方法的玉米茎秆组织结构切片质量,为研究玉米茎秆结构与其倒伏的关系奠定技术基础。结果表明:徒手切片法是获得成熟玉米秆组织结构切片较为方便、快速的方法,切片面积较大,适合大范围观察统计;冰冻切片法是获得成熟玉米秆组织结构较快的方法,切片面积较小,适合小范围观察;薄切片是获得高质量成熟玉米秆组织结构切片的最好方法,但切片面积太小,适合高倍数显微观察和小范围电镜扫描观察组织结构;石蜡切片不适合作为成熟玉米秆的组织切片方法。研究认为,徒手切片法是最适合成熟玉米秆组织结构观察研究的制片方法。     

The Use of Ethylenediamine in Softening Hard Plant Structures for Paraffin Sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.     

Preference for bedding material in Syrian hamsters

This study aimed to determine whether Syrian (golden) hamsters, Mesocricetus auratus, prefer certain bedding materials and whether bedding material can affect paw condition, body weight gain and wheel-running activity. In a first experiment, 26 male hamsters had access to two connected cages, each cage containing a different bedding material (either pine shavings, aspen shavings, corn cob or wood pellets). In a second experiment, 14 male hamsters had access to four connected cages that contained the different bedding materials and also a piece of paper towel to serve as nest material. In a third experiment, 30 male hamsters were each placed in a single cage, 10 of them with pine shavings, 10 with aspen shavings and 10 with corn cob, and they were monitored for 50 days. Significant preferences in the first experiment were: pine shavings over aspen shavings, corn cob over wood pellets, pine shavings over corn cob and aspen shavings over wood pellets (aspen shavings versus corn cob was not tested). However, there was no significant preference expressed in the second experiment, suggesting that the general preference for shavings in the first experiment was based on bedding material suitability as a nesting material. No significant effect of bedding material on paw condition, body weight gain and wheel-running activity was detected. None of the four bedding materials tested in this study can be judged to be inappropriate in the short term if nesting material is added to the cage and if the litter is changed regularly.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry.

A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

Utilisation of Toluidine blue O pH 4.0 and histochemical inferences in plant sections obtained by free-hand

Protoplasma - For the observation of plant material under a light microscope, free-hand sectioning is usually the fastest and most practical procedure. The staining of the obtained cuts is a...     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry

Summary A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

A Method to Identify Microinjected Nephrons of Rat

This paper describes a technique for identifying individual nephrons that have been subjected to micropuncture. The general location of the nephron is marked on the surface of the kidney by implanting two micropipette tips on opposite sides of it two or three tubule diameters away. The tubule itself is marked by the injection into the lumen of a tracer material, for purposes of this account, a suspension of E. coli. After perfusion fixation the kidneys are removed and a block of tissue containing the extrapapillary portion of the nephron is excised. This block is cut into thin slices parallel to the surface of the kidney; these are embedded in plastic for subsequent sectioning. On sectioning, the marker material makes the nephron in question readily discernible under the microscope. A major advantage of this technique is that it allows the tubule of interest to be located as much as 48 hours later.     

Phenotyping tree resistance to a bark‐chewing insect,the pine weevil Hylobius abietis

Breeding for resistance to forest pests and pathogens is emerging as a promising tool for minimising the impact of the increasing biotic threats that our forests are experiencing as a consequence of global change. Efficient phenotyping protocols of resistance are urgently needed. Here we present the results of two experiments aimed to determine whether the variation in resistance to the pine weevil Hylobius abietis, a harmful pest of European conifers, can be inferred by nondestructive bioassays using excised plant material collected in forest genetic trials. Weevil damage and amount of nonvolatile resin induced by weevil feeding were assessed in young trees and in branches of adult trees using several phenotyping procedures (bioassays using either living trees, excised plant material and cut stem twigs) on four pine species (Pinus pinaster, P. radiata, P. sylvestris and P. pinea). Half of the plants were previously induced with methyl jasmonate (MJ), a treatment that is known to affect resistance to the pine weevil. In Experiment 1, living and excised plants showed parallel results: MJ treatment significantly reduced weevil damage, and saplings responded to weevil damage locally increasing the nonvolatile resin (NVR) in the stems proportionally to the damage suffered. This response was, however, slightly lower in excised than in living saplings. On the contrary, patterns of weevil feeding on stem twigs completely departed from those observed in living and excised seedlings. Moreover, cut stem twigs were unable to respond to weevil feeding increasing NVR according to the weevil damage. In Experiment 2, assessment of weevil damage on excised branches explained around 50% of variation in damage on living branches. This relationship became much more pronounced (R² = 0.81) when explored at the mean treatment level; branch manipulation did not alter the patterns of variation in resistance across pine species or MJ treatments. Irrespective of the assessment procedure, MJ consistently decreased weevil damage in all pine species, with larger reduction in weevil damage in stone and maritime pine than in radiata and Scots pine. Radiata pine was the most resistant while Scots pine was the most susceptible to the pine weevil. Overall, results suggest that using excised plant material is an operative alternative for phenotyping weevil resistance whenever care is taken to maintain the functionality of the excised plant material. This will allow taking advantage of multiple available conifer genetic trials to deepen the ecological genetics of resistance to the pine weevil and to screen for resistance without compromising the long‐term utility of those genetic trials.     

A cryoultratomy method and its use in immunocytochemistry]

A method of frozen ultrathin sectioning for tissues and suspension material is presented. Practical aspects and results in the field of cryoultratomy are observed in detail. An example of a good preservation of tissue ultrastructure and application of cryosections to immunocytochemical investigations is given.     

Development and nature of the partition layer in Tilletia caries teliospore walls.

Developing Tilletia caries teliospores were studied with thin sectioning procedures. After the W1 and W2 spore walls are formed, lamellar material begins to form adjacent to the W2 wall layer. The patches of lamellar material become continuous, and additional layers are added. After the W3 wall starts to form, the lamellar material is difficult to see without special staining. The lamellar material makes it difficult to get resins to penetrate the partition layer of teliospore walls.     

Adaptation of a sliding microtome for sectioning of material embedded in epoxy resins]

The employment of a sliding microtome of sectioning plastic embedded material with glass knives is described. Using a new knife holder and a modificated device for fixing plastic blocks succeeded in cutting sections 1--10 micron thick of relatively large pieces of tissue.     

Orienting Minute Objects for Sectioning in a Definite Plane

Minute objects can be prepared for sectioning in a definite plane by a method which reembeds them directly on the cutting block under a dissecting microscope. By melting the paraffin immediately around the specimen, the latter can be oriented with reference to the planes of the block. After trimming, the block can be oriented squarely with reference to the microtome knife. Objects as small as 0.2 mm. have been cut successfully. The material sectioned included carpel primordia of Lathyrus, and young embryos, shoot apices and young axillary buds of Pinus. The technic is simpler than most methods previously suggested and it permits good control over the plane of sectioning.     

Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol

Utilization of ethanol produced from biomass has the potential to offset the use of gasoline and reduce CO(2) emissions. This could reduce the effects of global warming, one of which is the current outbreak of epidemic proportions of the mountain pine beetle (MPB) in British Columbia (BC), Canada. The result of this is increasing volumes of dead lodgepole pine with increasingly limited commercial uses. Bioconversion of lodgepole pine to ethanol using SO(2)-catalyzed steam explosion was investigated. The optimum pretreatment condition for this feedstock was determined to be 200 degrees C, 5 min, and 4% SO(2) (w/w). Simultaneous saccharification and fermentation (SSF) of this material provided an overall ethanol yield of 77% of the theoretical yield from raw material based on starting glucan, mannan, and galactan, which corresponds to 244 g ethanol/kg raw material within 30 h. Three conditions representing low (L), medium (M), and high (H) severity were also applied to healthy lodgepole pine. Although the M severity conditions of 200 degrees C, 5 min, and 4% SO(2) were sufficiently robust to pretreat healthy wood, the substrate produced from beetle-killed (BK) wood provided consistently higher ethanol yields after SSF than the other substrates tested. BK lodgepole pine appears to be an excellent candidate for efficient and productive bioconversion to ethanol.     

Applying Multiphoton Imaging to the Study of Membrane Dynamics in Living Cells

The endomembrane system of a cell is a highly dynamic, ephemeral structure that is difficult to visualize. Reconstructions from sections of fixed material can provide high-resolution information on intercellular membrane architecture, but such techniques are fraught with artifacts and are of little help in understanding the dynamics of intracellular membrane traffic. Recently, the availability of fluorescent membrane probes and the development of techniques for optically sectioning intact specimens have allowed glimpses of membrane dynamics to be visualized in living tissue. In this review we discuss the potential of a new optical sectioning technique, multiphoton imaging, for visualizing membrane dynamics in living cells. Multiphoton microscopy offers an unparalleled ability to obtain images from deep within specimens while minimizing the effects of phototoxicity.     

Light Microscopic Identification and Photography of Golgi Impregnated Central Nervous System Neurons During Sectioning for Electron Microscopy

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Light microscopic identification and photography of Golgi impregnated central nervous system neurons during sectioning for electron microscopy.

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin sections of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which part of the neuron and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

OBSERVATIONS ON FIBRILLOGENESIS IN THE CONNECTIVE TISSUE OF THE CHICK EMBRYO WITH THE AID OF SILVER IMPREGNATION

A technique is described which combines silver impregnation and ultrathin sectioning for the electron microscopic demonstration of fibrils in the connective tissue of the chick embryo. The electron micrographs presented in this paper provide evidence for the specificity and completeness of the silver-impregnation technique. It has been shown that, in this particular tissue after fixation in neutral formalin and at the stage of development represented by our material, the argyrophil fibers are embedded in a material which is continuous with the body of the fibroblasts.     

The genetics of shortleaf pine (<Emphasis Type="Italic">Pinus echinata</Emphasis> mill.) with implications for restoration and management

Shortleaf pine (Pinus echinata Mill.) is an important commercial timber resource and forest ecosystem component in the southeastern USA. The species occurs in mainly drier sites as an early- to mid-successional species, is fire-adapted, and it plays an important role in the fire ecology of the region. However, shortleaf pine genetics are not well-studied, especially in this era of molecular genetics and genomics. Most genetics research about the species has focused on provenance testing. Generally, shortleaf pine performs well in colder areas, when compared to loblolly pine (Pinus taeda L.), a close relative, which is faster growing and the most common plantation species in the region. Though not as advanced in genetic improvement as loblolly pine, tree breeders have improved shortleaf pine in one to two generations of selection, and diverse, genetically improved shortleaf pine materials are available to foresters and landowners throughout the southeastern USA. Researchers have also studied the genetic variation of shortleaf pine using various molecular markers and have found that shortleaf pine is generally a prolific outcrosser, a trait it shares with other non-isolated members of the family Pinaceae. In recent years, however, it has shared less genetic material across long ranges, probably because of habitat fragmentation. Various anthropogenic factors also affect shortleaf pine’s future, as recent studies show that shortleaf pine introgression with loblolly pine puts the species—and the resiliency of southeastern forests—at risk. Importantly, fire exclusion is a likely cause of the increase in introgression. Herein, we provide further details and up-to-date genetic information and resources for foresters and ecologists interested in the restoration and management of shortleaf pine.     

A Method for Embedding Thin Membranes in Historesin

A method is described for flat-embedding thin membranous tissues in Historesin. It allows easy orientation for sectioning large areas parallel to the surface. Selected fields can be monitored from the unfixed specimen, throughout preparation, to mounting on the microscope slide. For cross-sectioning, the flat-embedded tissue can be stacked and re-embedded to increase the amount of material examined per section.