Data reported in this work are part of the USDA-ARS National Program 306 Project 'Values-Added Products from Cottonseed' research supported by the Agency's in-house funding. Publication of this paper is supported in part by the Journal of Visualized Experiments. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. We acknowledge the constructive comments from JoVE science editor and peer reviewers in the review and revision process.

1. Cottonseed and Soy Meal-based Products (Figure 1)
<ol>
	<li>Obtain the raw materials, defatted cottonseed and soy meals, from commercially available sources.</li>
	<li>Obtain the working meal by grinding the solid defatted meal in a cyclone sample mill to pass a 0.5 mm steel screen16.</li>
	<li>Prepare water washed meals from the working meals after water extraction (25 g meal:200 ml water) to separate water soluble components in the meals21.</li>
	<li>Prepare protein isolates from the working meals by alkali extraction and acid precipitation16.</li>
</ol>
2. Preparation of Wood Veneer Strips
<ol>
	<li>Cut wood veneers (1.59 mm thick) obtained from a commercially available source into strips 25.4 mm wide by 88.9 mm long.</li>
	<li>Pencil mark a line across the wood grain at 25.4 mm (1.0&#34;) length from one end of each strip. Label these strips appropriately with testing treatments or numbers. 5 - 10 wood pairs are prepared for each testing variable.</li>
</ol>
3. Preparation of Adhesive Slurries
<ol>
	<li>Calculate the amount of water washed meal needed per the wood specimens for testing, by application rate (e.g., 4 mg dry content cm-2) x total bonding area (e.g., 581 cm2 of 90 wood strips with 2.54 x 2.54 cm bonding area each) plus about 30% extra for enoughness (i.e., 4 x 581 x 130% 3 g of water washed meal for the example).</li>
	<li>Mix water washed meal with deionized water (3:25 w/w), and stir with a magnetic stir bar for 2 hr in a beaker sealed with Parafilm.</li>
</ol>
4. Preparation of Bonded Wood Specimens
<ol>
	<li>Brush adhesive slurry onto one end of 2 wood veneer strips covering 25.4 mm (1.0&#34;) length. Air-dry for 10 - 15 min or until tacky.</li>
	<li>Brush a second layer of adhesive slurry on top of the first layer and air-dry again. The amount of dry adhesive preparation applied is about 4.5 mg dry solid per cm2 of bonding are of each wood strip.</li>
	<li>Overlap the tacky adhesive coated area (25.4 x 25.4 mm or 1.0&#34; x 1.0&#34;) of 2 wood veneer strips. Hot-press using a Benchtop Heated Press at 100 &#176;C for 20 min at a pressure of 400 psi (2.8 MPa). Note the pressure is the force applied by the press divided by the overlapped area of the wood samples. These bonding parameters may be changed as needed for each testing variable.</li>
	<li>Cool and condition the bonded wood specimens for 48 hr in a conditioning room or an incubator with humidity control (temperature of 22 - 23 &#176;C and relative humidity of 50 - 60%;&#160;Figure 2).</li>
</ol>
5. Water Resistance Experiments
<ol>
	<li>Immerse the bonded wood specimens, after initial conditioning, in tap water for 48 hr in a plastic tray at RT (22 - 23 &#176;C). The wet specimens after soaking are tested immediately for the shear strength at break and reported as wet strength. Excess water on the veneer surface may be removed by gently patting with paper tissue prior to measurements.</li>
	<li>Immerse another set of bonded wood specimens, after initial conditioning, in a water bath at 63 &#186;C for 4 hr, then dry at room conditions (temperature of 22 - 23 &#176;C and relative humidity of 50 - 60%) O/N (18 - 20 hr). Repeat the immersion-drying cycle once with a 48 hr drying time. The dried specimens are then tested for the shear strength at break and reported as soaked adhesive strength.</li>
</ol>
6. Lap Shear Strength Measurements
<ol>
	<li>Fit a bonded wood specimen into the 32 x 40 mm fishscale gridded wedge grips on a Materials Tester with a gripping pressure of 7 MPa, and set the crosshead speed at 1 mm min-1.</li>
	<li>Measure and record the shear strength at break for each bonded wood specimen. The results of multiple measurements are averaged for each adhesive formulation and test variable.</li>
</ol>

<ol>
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Soc. 72 (5), 507-510 (1995).</li><li>Li, K., Peshkova, S., Geng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+soy+protein-Kymene+adhesive+systems+for+wood+composites.">Investigation of soy protein-Kymene adhesive systems for wood composites.</a> J. Am. Oil Chem. Soc. 81 (5), 487-491 (2004).</li><li>Qi, G., Li, N., Wang, D., Sun, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+physicochemical+properties+of+soy+protein+modified+by+sodium+bisulfite.">Adhesion and physicochemical properties of soy protein modified by sodium bisulfite.</a> J. Am. Oil Chem. Soc. 90 (12), 1917-1926 (2013).</li><li>Cheng, H. N., Dowd, M. K., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+modified+cottonseed+protein+adhesives+for+wood+composites.">Investigation of modified cottonseed protein adhesives for wood composites.</a> Ind. Crop. Prod. 46, 399-403 (2013).</li><li>Pan, Z., Cathcart, A., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+and+chemical+treatments+to+improve+adhesive+property+of+rice+bran.">Thermal and chemical treatments to improve adhesive property of rice bran.</a> Ind. Crop. Prod. 22 (3), 233-240 (2005).</li><li>Nordqvist, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheat+gluten+fractions+as+wood+adhesives-glutenins+versus+gliadins.">Wheat gluten fractions as wood adhesives-glutenins versus gliadins.</a> J. Appl. Polymer Sci. 123 (3), 1530-1538 (2012).</li><li>Bandara, N., Chen, L., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+modified+triticale+distillers+grain+proteins.">Adhesive properties of modified triticale distillers grain proteins.</a> Int. J. Adhes. Adhes. 44, 122-129 (2013).</li><li>Li, N., Qi, G., Sun, X. S., Stamm, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+canola+oil+based+polyurethane+wood+adhesives.">Characterization of canola oil based polyurethane wood adhesives.</a> Int. J. Adhes. Adhes. 31 (6), 559-564 (2011).</li><li>Xiao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+sorghum+lignin+to+improve+adhesion+strength+of+soy+protein+adhesives+on+wood+veneer.">Utilization of sorghum lignin to improve adhesion strength of soy protein adhesives on wood veneer.</a> Ind. Crop. Prod. 50, 501-509 (2013).</li><li>Moubarik, A., Grimi, N., Boussetta, N., Pizzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+lignin+from+Moroccan+sugar+cane+bagasse:+Production+of+lignin-phenol-formaldehyde+wood+adhesive.">Isolation and characterization of lignin from Moroccan sugar cane bagasse: Production of lignin-phenol-formaldehyde wood adhesive.</a> Ind. Crop. Prod. 45, 296-302 (2013).</li><li>Patel, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+chitosan-based+adhesive.+Application+to+wood+bonding.">Development of a chitosan-based adhesive. Application to wood bonding.</a> J. Appl. Polymer Sci. 127 (6), 5014-5021 (2013).</li><li>He, Z., Cao, H., Cheng, H. N., Zou, H., Hunt, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+vigorous+blending+on+yield+and+quality+of+protein+isolates+extracted+from+cottonseed+and+soy+flours.">Effects of vigorous blending on yield and quality of protein isolates extracted from cottonseed and soy flours.</a> Modern Appl. Sci. 7 (10), 79-88 (2013).</li><li>Amico, S., Hrabalova, M., Muller, U., Berghofer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+of+spruce+wood+with+wheat+flour+glue-Effect+of+press+temperature+on+the+adhesive+bond+strength.">Bonding of spruce wood with wheat flour glue-Effect of press temperature on the adhesive bond strength.</a> Ind. Crop. Prod. 31, 255-260 (2010).</li><li>Gao, Q., Shi, S. 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The authors have nothing to disclose.

This paper presents a basic procedure to prepare and test plant seed-based products as wood adhesives. The adhesive slurries exampled in this protocol are simply the defatted seed meal product and water. Various adhesive formulations can be reached by addition of testing reagents (such as sodium dodecyl sulfate, sodium bisulfite or tung oil)5,6,23 and/or changes in mixing conditions (such as pH, ratio of solid and water)3,24,25. Adjustment of the adhesive formulation is also needed if the rheologica...

Adhesive bonding of wood plays an increasing role in the forest product industry and is a key factor for efficiently utilizing timber resources1. Interest in the use of natural product-based adhesives for wood increased steadily from the 1930s to reach a peak around 19602. After this period, the price of petroleum-based adhesives became so low that they displaced protein adhesives from several traditional markets. In the past two decades, this trend has reversed with renewed interest in the use of materials that are renewable, biodegradable, and more environmentally acceptable. These natural resources include, but are not limited to, soy protein3-5, cottonseed protein6, rice bran7, wheat gluten8, distillers grain protein9, canola protein and oil10-12, lignin from sorghum and sugar cane bagasse13,14, and polysaccharides derived from shrimp shells15.
Whereas seed protein isolates have been widely evaluated as potential wood adhesives, the isolation procedure involves corrosive alkaline and acidic reagents and it makes isolate-based adhesives relatively expensive and less environment-friendly16. Thus, some defatted seed meals (flours) with or without treatment have also been tested for the adhesive purpose, even though the adhesive properties of these meals do not perform as well as protein isolates17-19. We have sequentially fractionated cottonseed meal (CM) into different fractions, and examined their adhesive strength in bonding wood veneers20,21. The water-insoluble solid fraction (hereinafter washed cottonseed meal-WCM) could be used as wood adhesives, comparable to cottonseed protein isolate (CSPI), and would be less costly to prepare than CSPI.
Adhesive strength and water resistance are two critical parameters in evaluating the performance of a potential adhesive material. Here, the adhesive strength is reported as the shear strength at break of the lap bond of each wood specimen. Water resistance of the adhesive is measured by the change in lap shear strength of the bonded wood specimen at break due to water soaking. Using defatted cottonseed and soy meals as raw materials, this protocol provides a simple and straightforward way to prepare and test plant seed-based products as wood adhesives. This protocol would be helpful in facilitating the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Material and Equipment</td>
			<td style="width:227px;">Supplier/Manufacturer</td>
			<td style="width:227px;"></td>
			<td style="width:211px;">Address</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">defatted cottonseed meal</td>
			<td>Kentwood Co-op</td>
			<td></td>
			<td>Kentwood, LA, USA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defatted soy meal</td>
			<td>Kentwood Co-op</td>
			<td></td>
			<td>Kentwood, LA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">wood veneers</td>
			<td>Certainly Wood, Inc.</td>
			<td></td>
			<td>East Aurora, NY, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cyclone sample mill (model 3010-014)</td>
			<td>UDY Corporation</td>
			<td></td>
			<td>Fort Collins, CO, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop Heated Press&#160; (Model 3856)</td>
			<td>Carver, Inc.</td>
			<td></td>
			<td>Wabash, IN. USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials Tester</td>
			<td>Zwick GmbH &#38; Co.</td>
			<td></td>
			<td>Ulm, Germany</td>
		</tr>
	</tbody>
</table>

preparation and testing of plant seed meal-based wood adhesives

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable and environment-friendly. These natural products may serve as alternatives to petroleum-based adhesives to ease environmental and sustainability concerns. This work demonstrates the preparation and testing of the plant seed-based wood adhesives using cottonseed and soy meal as raw materials. In addition to untreated meals, water washed meals and protein isolates are prepared and tested. Adhesive slurries are prepared by mixing a freeze-dried meal product with deionized water (3:25 w/w) for 2 hr. Each adhesive preparation is applied to one end of 2 wood veneer strips using a brush. The tacky adhesive coated areas of the wood veneer strips are lapped and glued by hot-pressing. Adhesive strength is reported as the shear strength of the bonded wood specimen at break. Water resistance of the adhesives is measured by the change in shear strength of the bonded wood specimens at break after water soaking. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for substitution of synthetic-based wood adhesives. Adjustments to the adhesive formulation with or without additives and bonding conditions could optimize their adhesive properties for various practical applications.

Each adhesive formulation&#8217;s performance is determined by the shear strength of the bonded wood specimen at break and the values vary depending on the dimensions of the wood veneer used. For example, in Table 1, the dry and soaked adhesive strength values of the bonded specimens are lower when thinner and narrower maple strips are used (see Cottonseed-1), as opposed to the thicker and wider strips of Cottonseed-2 recommended in the protocol, using the same cottonseed-based adhesive formulation. Also...

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Preparation and Testing of Plant Seed Meal-based Wood Adhesives

To facilitate the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives, this work demonstrates the preparation and testing of plant seed-based wood adhesives. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for the substitution of synthetic-based wood adhesives.

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable ...

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8.6K Views.  United States Department of Agriculture. To facilitate the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives, this work demonstrates the preparation and testing of plant seed-based wood adhesives. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for the substitution of synthetic-based wood adhesives.

Video: Preparation and Testing of Plant Seed Meal-based Wood Adhesives

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking.
The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink&#39;s pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.

Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for plastic materials to be re-utilized, they must be identified and separated according to their polymer composition. In this study, the identification of these materials was performed using near-infrared spectroscopy (NIR), which identified material based on their physical-chemical properties. Advantages of the NIR method are a low environmental impact and rapid measurement (within a few seconds) in the spectral range of 1600-2400 nm without special sample preparation. Limitations include its inability to analyze dark materials. The identified polymers were utilized as a component for wood-polymer composite (WPC) that consists of a polymer matrix, low cost fillers, and additives. The components were first compounded with an agglomeration apparatus, followed by production by extrusion. In the agglomeration process, the aim was to compound all materials to produce uniformly distributed and granulated materials as pellets. During the agglomeration process, the polymer (matrix) was melted and fillers and other additives were then mixed into the melted polymer, being ready for the extrusion process. In the extrusion method, heat and shear forces were applied to a material within the barrel of a conical counter-rotating twin-screw type extruder, which reduces the risk of burning the materials and lower shear mixing. The heated and sheared mixture was then conveyed through a die to give the product the desired shape. The above-described protocol proved the potential for re-utilization of CDW materials. Functional properties must be verified according to the standardized tests, such as flexural, tensile, and impact strength tests for the material.

Secondary material streams have been shown to include potential raw materials for production. Presented here is a protocol in which CDW-plastic waste as a raw material is identified, followed by various processing steps (agglomeration, extrusion). As a result, a composite material was produced, and mechanical properties were analyzed.

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...