We would like to thank the financial support provided by Worrell Water Technologies, LLC. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture.

1. Measurement of Gaseous ClO2 in the Headspace of a Closed Chamber
<ol>
	<li>Obtain the materials: ClO2 pouch (0.5 g of ClO2 slurry (9.5% a.i.) in a polymer film selected for its release rate (total surface area of 6 cm2); the exact components are proprietary), a glass chamber (19.14 L), and a lid with switchable gas inlet and outlet.</li>
	<li>Attach the ClO2 pouch to the lid using double-sided tape.</li>
	<li>Close the chamber by sealing the lid with petroleum jelly.</li>
	<li>Connect the inlet and outlet of a ClO2 gas detector to the chamber. 
	NOTE: This is a gas circulation system, and no gas loss occurred when taking measurements.</li>
	<li>Switch on the inlet and outlet gas flow and measure the ClO2 concentration in the chamber after incubating for 0, 1, 2, 3, 4, 24, 26, 28, and 48 h.</li>
	<li>Monitor the temperature and relative humidity (RH) in the chamber with temperature and RH data loggers.</li>
</ol>
2. Fruit Preparation and Storage
<ol>
	<li>Obtain 15 kg of fresh grape tomatoes (Solanum lycopersicum var. cerasiforme) from a local retailer. Ensure that the fruits are healthy and have no visual flaws.</li>
	<li>Preparation of inoculum
	<ol>
		<li>Use strains of E. coli (wild type) and A. alternata from citrus fruit surfaces27 for inoculation.</li>
		<li>Culture E. coli on E. coli agar (ECA) at 35 &#176;C for 1 day27 and then re-culture the organisms on a new plate for 1 day. Confirm the organisms by sampling the ECA plates with a bac-loop, streaking the bacteria on Levine eosin methylene blue (EMB) agar, and incubating for 24 h at 35 &#176;C; cultures that turn reflective, metallic green are positive for E. coli.</li>
		<li>Culture A. alternata on potato dextrose agar (PDA) at 25 &#176;C until spores appear.</li>
		<li>Scrape the E. coli cells from the agar plate into 50 mL of sterile distilled water until the estimated concentration reaches 9 log CFU/mL using a comparison with McFarland equivalence turbidity standards. Add 1,950 mL of sterile water containing 0.1% Tween-20 to make 2 L total of the final inoculum.</li>
		<li>Verify the cell concentration by dilution plating on EC agar plates. Scrape the A. alternata spores from the culture medium and suspend them into 2 L of sterile distilled water containing 0.1% Tween-20. 
		NOTE: The final E. coli population was 7.5 log CFU/g, and the A. alternata population was 5.5 log CFU/g.</li>
	</ol>
	</li>
	<li>Place 7 kg of the tomatoes into a 10 L stainless steel pan that is completely covered by an autoclavable bag. Place the bag and pan in a safety hood. Apply the inoculum solution (2 L) to the fruits using a trigger sprayer applied from the top while gently stirring the fruits with a gloved hand.
	<ol>
		<li>After 5 min, place the tomatoes in a single layer on sterilized sheets and allow them to air dry for 2 h. Put about 200 g of fruit each into twent-four 1 lb (~1.14 L) perforated clamshells.</li>
	</ol>
	</li>
	<li>Carefully fold the contaminated foils and place them into the steel pan. Remove the gloves and put them into the pan. Wrap the autoclavable bag and autoclave all contaminated supplies at 121 &#176;C for 25 min.</li>
	<li>Attach ClO2 pouches to the lids of 12 clamshells. Use the other 12 clamshells as controls. Weigh each whole clamshell. Store the fruit at 20 &#176;C for 14 days.</li>
	<li>Take samples on days 3, 7, 10, and 14. Sample three clamshells, representing 3 replicates, per treatment per day.</li>
</ol>
3. Monitoring of ClO2 Concentration in the Clamshells
<ol>
	<li>Insert the inlet and outlet tubing of the ClO2 gas detector into the center of the clamshells, with a 2 cm distance between the two ends, and take the ClO2 measurement on days 3, 7, 10, and 14.</li>
</ol>
4. Determination of Microbial Population and Fruit Quality Attributes
<ol>
	<li>Agitate 5 fruits (about 60 g) from each replicate at 100 rpm for 1 h in a sterilized sampling bag along with 99 mL of sterile potassium phosphate buffer (0.01 M, pH 7.2) on an orbital shaker.
	<ol>
		<li>Plate serial dilutions (1-, 10-, and 100-fold) of the buffer wash, 50 &#181;L each, on ECA (for E. coli) and PDA (for A. alternata) using a spiral plater.</li>
		<li>Incubate the ECA plates at 35 &#176;C for 24 h and the PDA plates at 25 &#176;C for 3 days. Read the microbial colony count using an optical plate reader. Sanitize all equipment which contacted the contaminated fruit after use.</li>
	</ol>
	</li>
	<li>Measure fruit firmness with a fruit firmness tester using the manufacturer&#39;s protocol. Calibrate the tester before each use. Measure 20 fruit for each replicate and express the results as the pressure force, Newton (N), required to compress the fruit by 1 mm (converted to N&#183;m&#8722;1).</li>
	<li>Weigh the whole clamshell with the fruit at the beginning of and during storage and calculate the weight loss in comparison to the initial weight.</li>
</ol>
5. Statistical Analysis
<ol>
	<li>Replicate all experiments in triplicate. Analyze the data using analysis of variance (ANOVA). Determine the mean separation by Duncan&#39;s multiple range test; the significance is defined at p &#60;0.05.</li>
</ol>

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The authors declare that they have no competing financial interests.

Chlorine dioxide is an ideal biocide to prevent food decay. However, it is unstable at high concentrations and non-transportable, requiring costly generators or inefficient two-part powder mixing. This study examined the application of a stable, ready-to-use form of chlorine dioxide to reduce food spoilage and the incidence of foodborne illness. In contrast to other chlorine dioxide application technologies currently in use, the commercial ClO2 used here is cost effective, has a long shelf life, and does not r...

A diet rich in fresh fruits and vegetables may help to reduce the risk of many diseases, including coronary heart disease and specific types of cancers1. However, there are a number of foodborne microbial pathogens, such as Escherichia coli, Salmonella enterica, and Listeria monocytogenes, associated with the consumption of fresh fruits and vegetables that can cause illness or even death among consumers who eat contaminated produce2. For example, E. coli O157:H7 outbreaks have been associated with grapes, tomatoes, and strawberries3,4, and hepatitis A outbreaks have been associated with fresh blueberries5. In addition, microbial contamination can cause substantial product loss through postharvest decay6. Alternaria alternata is an important plant pathogenic fungus that is known to cause leaf spots and other diseases in over 380 host species of plants7. It has been shown to be the cause of an Alternaria black spot8, a stem canker disease and a leaf blight of tomatoes9. Therefore, a safe and effective postharvest decontamination treatment is needed to both control foodborne pathogens and to prevent postharvest decay in fresh produce.
Low- and non-residue technologies are new trends for alternative sanitizers. A variety of postharvest fungicides have been used to reduce spoilage organisms and to prevent foodborne illness. Ozone, a strong antimicrobial agent, has been shown to preserve the quality and freshness of strawberries and blueberries10,11. However, ozone may cause oxidation of fruit surface tissue and can result in discoloration and the deterioration of flavor quality12. Chlorine has been used to sanitize fresh produce, such as blueberries and apples13. While effective, chlorine can react with nitrogen-containing compounds or ammonia, resulting in carcinogenic byproducts14, especially when used for the sanitization of fresh fruit15.
Chlorine dioxide (ClO2), an alternative sanitizer, was approved by both China and the US for the postharvest treatment of fruits and vegetables16. ClO2 is a water-soluble oxidizing agent with an oxidation capacity 2.5 times greater than that of free chlorine17. ClO2 is highly effective at low concentrations and with a short contact time18. ClO2 has low toxicity and minimal corrosiveness at the concentrations used for disinfection, and it is recognized as one of the most effective bactericidal and fungicidal agents for use in a variety of settings19,20,21.
Numerous research results have shown that ClO2 can control foodborne pathogens and postharvest decay16. For example, ClO2 gas has been used to inactivate L. monocytogenes, Salmonella, and E. coli O157:H7 and to prevent blueberry and strawberry spoilage22,23. ClO2 gas reduces the risk of microbial contamination while maintaining the attributes of fresh fruit, and it was effective at controlling the postharvest decay of strawberries24. However, it is unstable at high concentrations and non-transportable, historically requiring costly generators on site or inefficient two-part powder mixing.
However, a new ClO2 product with a ready-made, controlled-release formulation (i.e., it does not require a generator or the premixing of ingredients) has been shown to be highly effective at controlling food spoilage organisms and pathogens in preliminary experiments25. It is a safe, cost-effective, non-corrosive, easily transportable, and controlled-release form of ClO2, with no adverse effects on the environment. Previous experiments have demonstrated that this slow-release ClO2 powder wrapped in filtration material and placed in clamshell packaging significantly reduced the decay of fresh blueberries and strawberries, decreased berry water loss, and maintained fruit firmness during postharvest storage25,26. Recently, a controlled-release ClO2 packet was developed by sealing a slurry form of ClO2 in a semipermeable polymer film. The objectives of this work were to: 1) monitor ClO2 gas release properties in both a closed container and in perforated clamshells, 2) investigate the effect of a controlled-release ClO2 pouch enclosed in a container on foodborne pathogens and the decay of grape tomatoes, and 3) evaluate the effects of the controlled-release ClO2 on the storage quality of grape tomatoes.

<table><tbody><tr><td>Curoxin&lt;sup&gt;&amp;#174;&lt;/sup&gt; chlorine dioxide pouch</td><td>Worrell Water Technologies</td><td></td><td>Slurry, a.i. 9.5% in sealed semi-permeable polymer film</td></tr><tr><td>Grape tomato</td><td>Santa Sweets, Inc</td><td></td><td>Santa Sweets Authentic&amp;nbsp;</td></tr><tr><td>ClO&lt;sub&gt;2&lt;/sub&gt; gas detector</td><td>Analytical Technology, Inc., Collegeville, PA</td><td></td><td>PortaSens II&amp;nbsp;</td></tr><tr><td>Perforated clamshell</td><td>Packaging Plus LLC, Yakima, WA</td><td></td><td>OSU #1, 1 lb</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;&amp;nbsp;</td><td></td><td></td><td>Wild Type (WT) from fruit surface</td></tr><tr><td>Alternaria alternata</td><td></td><td></td><td>from fruit surface</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; agar&amp;nbsp;</td><td>EC Broth, Oxoid, UK</td><td></td><td>EC Broth with 1.5% agar</td></tr><tr><td>Potato dextrose agar&amp;nbsp;</td><td>BD Difco, Sparks, MD</td><td></td><td></td></tr><tr><td>Levine eosin methylene blue agar</td><td>BD Difco, Sparks, MD</td><td></td><td></td></tr><tr><td>Trigger spray bottle&amp;nbsp;</td><td>Impact Products, LLC., Toledo, OH</td><td></td><td></td></tr><tr><td>Sterilized sampling bag&amp;nbsp;</td><td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td><td></td><td></td></tr><tr><td>Orbit shaker&amp;nbsp;</td><td>New Brunswick Scientific, New Brunswick, NJ</td><td></td><td>Innova 2100</td></tr><tr><td>IUL Instruments Neutec Eddy jet spiral plater inoculation plating system</td><td>Neutec Group Inc., Farmingdale, NY</td><td></td><td></td></tr><tr><td>EZ micro optical plate reader&amp;nbsp;</td><td>Synoptics, Ltd., Cambridge, UK</td><td></td><td>ProtoCOL</td></tr><tr><td>Fruit firmness tester&amp;nbsp;</td><td>Bioworks Inc, Wamego, KS</td><td></td><td>FirmTech 2&amp;nbsp;</td></tr><tr><td>Tinytag temperature and RH data logger</td><td>Gemini Data Loggers, West Sussex, UK</td><td></td><td></td></tr><tr><td>McFarland equivalence turbidity standard</td><td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td><td></td><td></td></tr></tbody></table>

controlled-release of chlorine dioxide in a perforated packaging system to extend the storage life and improve the safety of grape tomatoes

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties of the pouch were monitored in containers with or without fruit. The pouch was affixed to the inside of a perforated clamshell containing grape tomatoes, and the effect on microbial population, firmness, and weight loss was evaluated during a 14 day storage period at 20 &#176;C. Within 3 days, the ClO2 concentration in the clamshells reached 3.5 ppm and remained constant until day 10. Thereafter, it decreased to 2 ppm by day 14. The ClO2 pouch exhibited strong antimicrobial activity, reducing Escherichia coli populations by 3.08 log CFU/g and Alternaria alternata populations by 2.85 log CFU/g after 14 days of storage. The ClO2 treatment also reduced softening and weight loss and extended the overall shelf life of the tomatoes. Our results suggest that ClO2 treatment is useful for extending the shelf life and improving the microbial safety of tomatoes during storage without impairing their quality.

The release of ClO2 exhibited a linear pattern over the first few hours. The concentration increased about 2.38 ppm/h over the first 4 h. The release speed slowed after 24 h of incubation, and the ClO2 concentration reached 25.4 ppm. However, the concentration tended to be stable after 24 h of incubation (Figure 1).
The headspace ClO2 concentration in the clamshell with grape tom...

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Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties ...

controlled-release-chlorine-dioxide-perforated-packaging-system-to

Research

JoVE Journal

Environment

11.0K Views.  USDA, ARS, USHRL. Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

Video: Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Biochemistry

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

Bioengineering

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Immunology and Infection

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages.
Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

Developmental Biology

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

Medicine

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Biology

Molecule Delivery in Plants Using a Direct Infusion Device

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

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