NASA Exobiology grant 80NSSC19K0477 funded this research. We thank William Waite and Nicolas Espinoza for valuable discussions.

<ol>
	<li>Bohrmann, G., Torres, M. E., Schulz, H. D., Zabel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+hydrates+in+marine+sediments.">Gas hydrates in marine sediments.</a> Marine Geochemistry. , 481-512 (2006).</li><li>Ruppel, C. D., Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+climate+change+and+methane+hydrates.">The interaction of climate change and methane hydrates.</a> Reviews of Geophysics. 55 (1), 126-168 (2017).</li><li>Hammerschmidt, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+gas+hydrates+in+natural+gas+transmission+lines.">Formation of gas hydrates in natural gas transmission lines.</a> Industrial and Engineering Chemistry. 26, 851-855 (1934).</li><li>Ke, W., Kelland, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+hydrate+inhibitor+studies+for+gas+hydrate+systems:+a+review+of+experimental+equipment+and+test+methods.">Kinetic hydrate inhibitor studies for gas hydrate systems: a review of experimental equipment and test methods.</a> Energy &amp; Fuels. 30 (12), 10015-10028 (2016).</li><li>Kelland, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+kinetic+hydrate+inhibitors+from+an+environmental+perspective.">A review of kinetic hydrate inhibitors from an environmental perspective.</a> Energy &amp; Fuels. 32 (12), 12001-12012 (2018).</li><li>Walker, V. 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=Antifreeze+proteins+as+gas+hydrate+inhibitors.">Antifreeze proteins as gas hydrate inhibitors.</a> Canadian Journal of Chemistry. 93 (8), 839-849 (2015).</li><li>Bruusgaard, H., Lessard, L. D., Servio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+study+of+structure+I+methane+hydrate+formation+and+decomposition+of+water+droplets+in+the+presence+of+biological+and+polymeric+kinetic+inhibitors.">Morphology study of structure I methane hydrate formation and decomposition of water droplets in the presence of biological and polymeric kinetic inhibitors.</a> Crystal Growth &amp; Design. 9 (7), 3014-3023 (2009).</li><li>Jung, J. W., Espinoza, D. N., Santamarina, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+phenomena+relevant+to+CH4-CO2+replacement+in+hydrate-bearing+sediments.">Properties and phenomena relevant to CH4-CO2 replacement in hydrate-bearing sediments.</a> Journal of Geophysical Research. 115 (10102), 1-16 (2010).</li><li>Chen, X., Espinoza, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostwald+ripening+changes+the+pore+habit+and+spatial+variability+of+clathrate+hydrate.">Ostwald ripening changes the pore habit and spatial variability of clathrate hydrate.</a> Fuel. 214, 614-622 (2018).</li><li>DuQuesnay, J. R., Diaz Posada, M. C., Beltran, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+gas+hydrate+reactor+design:+3-in-1+assessment+of+phase+equilibria,+morphology+and+kinetics.">Novel gas hydrate reactor design: 3-in-1 assessment of phase equilibria, morphology and kinetics.</a> Fluid Phase Equilibria. 413, 148-157 (2016).</li><li>Udegbunam, L. U., DuQuesnay, J. R., Osorio, L., Walker, V. K., Beltran, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+equilibria,+kinetics+and+morphology+of+methane+hydrate+inhibited+by+antifreeze+proteins:+application+of+a+novel+3-in-1+method.">Phase equilibria, kinetics and morphology of methane hydrate inhibited by antifreeze proteins: application of a novel 3-in-1 method.</a> The Journal of Chemical Thermodynamics. 117, 155-163 (2018).</li><li>Espinoza, D. N., Santamarina, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-CO2-mineral+systems:+Interfacial+tension,+contact+angle,+and+diffusion+-+Implications+to+CO2+geological+storage.">Water-CO2-mineral systems: Interfacial tension, contact angle, and diffusion - Implications to CO2 geological storage.</a> Water Resources Research. 46 (7537), 1-10 (2010).</li><li>Sloan, E. D., Koh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clathrate Hydrates of Natural Gases. 3rd edn. , (2007).</li><li>Makogon, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydrates of natural gas. , 125 (1981).</li><li>Johnson, A. M., 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=Mainly+on+the+plane:+deep+subsurface+bacterial+proteins+bind+and+alter+clathrate+structure.">Mainly on the plane: deep subsurface bacterial proteins bind and alter clathrate structure.</a> Crystal Growth &amp; Design. 20 (10), 6290-6295 (2020).</li></ol>

There are no competing financial interests.

We have developed a method to form methane hydrate shells on sessile water droplets safely and share this method to machine and assemble a pressure cell rated to 10 MPa working pressure, as well as the pressurizing and cooling systems. The pressure cell is outfitted with a stage for the droplet containing embedded thermocouples, a sapphire window for visualizing the droplet, and a pressure transducer fixed to the top of the cell. The cooling system includes chilled ethylene glycol circulating through copper coils in a ta...

Gas hydrates are cages of hydrogen-bonded water molecules that trap guest gas molecules via van der Waals interactions. Methane hydrates form under high-pressure and low-temperature conditions, which occur in nature in the subsurface sediment along continental&#160;margins, under Arctic permafrost, and on other planetary bodies in the solar system1. Gas hydrates store several thousand gigatons of carbon, with important implications for climate and energy2. Gas hydrates can also be hazardous in the natural gas industry because conditions favorable for hydrates occur in gas pipelines, which can clog the pipes leading to fatal explosions and oil spills3.
Due to the difficulty of studying gas hydrates in situ, laboratory experiments are often employed to characterize hydrate properties and the influence of inhibitors and substrates4. These laboratory experiments are performed by growing gas hydrate at elevated pressure in cells of various shapes and sizes. Efforts to prevent gas hydrate formation in gas pipelines have led to the discovery of several chemical and biological gas hydrate inhibitors, including antifreeze proteins (AFPs), surfactants, amino acids, and polyvinylpyrrolidone (PVP)5,6. To determine the effects of these compounds on gas hydrate properties, these experiments have employed diverse vessel designs, including autoclaves, crystallizers, stirred reactors, and rocking cells, which support volumes from 0.2 to 106 cubic centimeters4.
The sessile droplet method used here and in previous studies7,8,9,10,11,12 involves forming a gas hydrate film on a sessile droplet of water inside a pressure cell. These vessels are made of stainless steel and sapphire to accommodate pressures up to 10-20 MPa. The cell is connected to a methane gas cylinder. Two of these studies used the droplet method to test AFPs as gas hydrate inhibitors compared to commercial kinetic hydrate inhibitors (KHIs), such as PVP7,11. Bruusgard et al.7 focused on the morphologic influence of inhibitors and found that droplets containing Type I AFPs have a smoother, glassy surface than the dendritic droplet surface without inhibitors at high driving forces.
Udegbunam et al.11 used a method developed to assess KHIs in a previous study10, which allows for the analysis of morphology/growth mechanisms, the hydrate-liquid-vapor equilibrium temperature/pressure, and kinetics as a function of temperature. Jung et al. studied CH4-CO2 replacement by flooding the cell with CO2 after forming a CH4 hydrate shell8. Chen et al. observed Ostwald ripening as the hydrate shell forms9. Espinoza et al. studied CO2 hydrate shells on various mineral substrates12. The droplet method is a relatively simple and cheap method to determine the morphologic effect of various compounds and substrates on gas hydrates and requires small amounts of additives due to the small volume. This paper describes a method for forming such hydrate shells on a droplet of water using a stainless-steel cell with a sapphire window for visualization, rated up to 10 MPa working pressure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CAMERA AND LAPTOP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera Body</td>
			<td>Nikon</td>
			<td>D7200</td>
			<td>Name in Protocol: camera</td>
		</tr>
		<tr>
			<td>Camera Control Pro 2 Software</td>
			<td>Nikon</td>
			<td></td>
			<td>Name in Protocol: camera software</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>HP Pavilion</td>
			<td>hp-pavilion-laptop-14-ce0068st</td>
			<td>Needs to be PC with plenty of storage (~ 1 Tb) 
			Name in Protocol: laptop</td>
		</tr>
		<tr>
			<td>Macrophotography Lens</td>
			<td>Nikon</td>
			<td>AF-S MICRO 105mm f/2.8G IF-ED Lens</td>
			<td>Name in Protocol: lens</td>
		</tr>
		<tr>
			<td>CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: DI water</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>VWR or grocery store</td>
			<td></td>
			<td>Buy just before nucleation 
			Name in Protocol: dry ice</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: ethanol</td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: ethylene glycol</td>
		</tr>
		<tr>
			<td>COOLING SYSTEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 in. O.D. x 3/8 in. I.D. x 25 ft. Polyethylene Tubing</td>
			<td>Everbilt</td>
			<td>Model # 301844</td>
			<td>For circulating coolant from chiller to copper coils in aquarium 
			Name in Protocol: 3/8&#8221; (inner diameter) plastic tubing</td>
		</tr>
		<tr>
			<td>Circulating chiller</td>
			<td>Polyscience</td>
			<td></td>
			<td>Name in Protocol: chiller</td>
		</tr>
		<tr>
			<td>Economical Flexible Polyethylene Foam Pipe Insulation</td>
			<td>McMaster-Carr</td>
			<td>4530K162</td>
			<td>3/4&#34; thick wall; 1/2&#34; inner diameter; R Value 3; 6&#39; long 
			Name in Protocol: foam pipe insulation</td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td>use any tubing that fits the airline connection in the lab and long enough to travel from the airline connection to the front of the aquarium</td>
		</tr>
		<tr>
			<td>DATALOGGER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Armature Multiplexer Module for 34970A/ 
			34972A, 20-Channel</td>
			<td>Keysight Technologies</td>
			<td>34901A</td>
			<td>Name in Protocol: datalogger multichannel</td>
		</tr>
		<tr>
			<td>Benchvue or Benchlink software</td>
			<td>Benchvue or Benchlink</td>
			<td></td>
			<td>Name in Protocol: temperature transducer software</td>
		</tr>
		<tr>
			<td>Data Acquisition/Switch Unit. GPIB, RS232</td>
			<td>Keysight Technologies</td>
			<td>34970A</td>
			<td>Name in Protocol: datalogger</td>
		</tr>
		<tr>
			<td>USB/GPIB interface</td>
			<td>Keysight Technologies</td>
			<td>82357B</td>
			<td>Name in Protocol: datalogger USB</td>
		</tr>
		<tr>
			<td>datalogger multichannel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schott Fostec -Llc 20510 Ace Fiber Optic Light Source</td>
			<td>Schott Fostec</td>
			<td>A20500</td>
			<td>3115PS-12W-B20 115 V ~AC 50/60Hz 5/4.5 W 
			Name in Protocol: light source unit</td>
		</tr>
		<tr>
			<td>Schott Fostec light source guide - single bundle</td>
			<td>Schott Fostec</td>
			<td>A08031.40</td>
			<td>Name in Protocol: fiber optic light source cable</td>
		</tr>
		<tr>
			<td>METHANE GAS AND REGULATOR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 OD in. x 20 ft. Copper Soft Refrigeration Coil</td>
			<td>Everbilt</td>
			<td>Model # D 04020PS</td>
			<td>For pressurizing ISCO pressure pump. An additional pack is needed for coolant circulation, as listed below. 
			Name in Protocol: high pressure-rated 1/4&#8221; copper pipe</td>
		</tr>
		<tr>
			<td>Methane cylinder regulator</td>
			<td>Airgas</td>
			<td>Y11N114G350-AG</td>
			<td>Name in Protocol: methane cylinder regulator</td>
		</tr>
		<tr>
			<td>Methane gas cylinder</td>
			<td>Airgas</td>
			<td>ME UHP300</td>
			<td>Name in Protocol: methane gas cylinder</td>
		</tr>
		<tr>
			<td>PRESSURE PUMP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in.&#160; flexible tubing, ~ 3 ft.</td>
			<td></td>
			<td></td>
			<td>Connect to pump inlet for leak test 
			Name in Protocol: 1/4&#34;&#160; flexible tubing</td>
		</tr>
		<tr>
			<td>260D Syringe Pump W/Controller</td>
			<td>Teledyne Instruments Inc.</td>
			<td>67-1240-520</td>
			<td>Name in Protocol: pressure pump</td>
		</tr>
		<tr>
			<td>Controller &#8722; Ethernet/USB</td>
			<td>Teledyne Instruments Inc.</td>
			<td>62-1240-114</td>
			<td>Purchase if you would like to install Labview onto computer and control pressure pump remotely. We did not do this.</td>
		</tr>
		<tr>
			<td>Smooth-Bore Seamless 316 Stainless Steel Tubing, 1/4&#34; OD, 0.035&#34; Wall Thickness, 1 Foot Long (x5)</td>
			<td>McMaster-Carr</td>
			<td>89785K824</td>
			<td>Name in Protocol: 1/4&#34; pipe</td>
		</tr>
		<tr>
			<td>Smooth-Bore Seamless 316 Stainless Steel Tubing, 1/8&#34; OD, 0.02&#34; Wall Thickness, 1 Foot Long (x4)</td>
			<td>McMaster-Carr</td>
			<td>89785K811</td>
			<td>Name in Protocol: 1/8&#34; pipe</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Reducing Union, 1/4 in. x 1/8 in. Tube OD (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-6-2</td>
			<td>Name in Protocol: 1/8&#8221; to 1/4&#8221; adapter</td>
		</tr>
		<tr>
			<td>PRESSURE CELL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>316 Stainless Steel Nut and Ferrule Set (1 Nut/1 Front Ferrule/1 Back Ferrule) for 1/4 in. Tube Fitting (20)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-NFSET</td>
			<td>Used for fitting connections where necessary 
			Name in Protocol: ferrule set</td>
		</tr>
		<tr>
			<td>316L Stainless Steel Convoluted (FM) Hose, 1/4 in., 316L Stainless Steel Braid, 1/4 in. Tube Adapters, 60 in. (1.5 m) Length</td>
			<td>Swagelok</td>
			<td>SS-FM4TA4TA4-60</td>
			<td>Connects pressure pump to pressure cell 
			Name in Protocol: 1/4&#34; braided stainless steel flexible pressure-rated hose</td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>ABAQUS FEA</td>
			<td></td>
			<td>Name in Protocol: simulation software</td>
		</tr>
		<tr>
			<td>Abrasion-Resistant Cushioning Washer for 7/8&#34; Screw Size, 0.875&#34; ID, 2.25&#34; OD, packs of 10 (x1)</td>
			<td>McMaster-Carr</td>
			<td>90131A107</td>
			<td>Name in Protocol: 2.25&#34; rubber washer</td>
		</tr>
		<tr>
			<td>Abrasion-Resistant Sealing Washer, Aramid Fabric/Buna-N Rubber, 3/8&#34; Screw Size, 0.625&#34; OD, packs of 10 (x1)</td>
			<td>McMaster-Carr</td>
			<td>93303A105</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>Acrylic Sheet | White 2447 / WRT31 
			Extruded Paper-Masked (Translucent 55% (0.118 x 12 x 12)</td>
			<td>Interstate Plastics</td>
			<td>ACRW7EPSH</td>
			<td>Machine a circle of acrylic to fit in the inner chamber of the pressure cell to serve as the background for imaging 
			Name in Protocol: acrylic disc</td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>AutoCAD</td>
			<td></td>
			<td>Name in Protocol: engineering design software</td>
		</tr>
		<tr>
			<td>Conax fitting</td>
			<td>Conax Technologies</td>
			<td>311401-011</td>
			<td>TG(PTM2/)-24-A6-T, OPTIONAL 1/4&#34; NPT 
			Name in Protocol: pressure seal connector</td>
		</tr>
		<tr>
			<td>High Accuracy Oil Filled Pressure 
			Transducers/Transmitters for General 
			industrial applications (x2)</td>
			<td>Omega Engineering, Inc.</td>
			<td>PX409-3.5KGUSBH</td>
			<td>Buy two so there is a backup. 
			Name in Protocol: pressure transducer</td>
		</tr>
		<tr>
			<td>HIGH PRESSURE CHAMBER&#160; PARTS</td>
			<td>Wither Tool, Die and Manufacturing Company</td>
			<td></td>
			<td>Machining for pressure cell parts as listed in CAD drawings (Figure S1) 
			Name in Protocol: Part B = stainless steel washer</td>
		</tr>
		<tr>
			<td>High-Strength 316 Stainless Steel Socket Head Screw, M5 x 0.80 mm Thread, 14 mm Long (x20)</td>
			<td>McMaster-Carr</td>
			<td>90037A119</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>High-Strength 316 Stainless Steel Socket Head Screw, M8 x 1.25 mm Thread, 25 mm Long (x20)</td>
			<td>McMaster-Carr</td>
			<td>90037A133</td>
			<td>Name in Protocol: M8 stainless steel screws</td>
		</tr>
		<tr>
			<td>Oil-Resistant Hard Buna-N O-Ring, 3/32 Fractional Width, Dash Number 120, packs of 50 (x1)</td>
			<td>McMaster-Carr</td>
			<td>5308T178</td>
			<td>Name in Protocol: 1&#34; o-ring</td>
		</tr>
		<tr>
			<td>Oil-Resistant Hard Buna-N O-Ring, 3/32 Fractional Width, Dash Number 128, packs of 50 (x1)</td>
			<td>McMaster-Carr</td>
			<td>5308T186</td>
			<td>Name in Protocol: 1.5&#34; o-ring</td>
		</tr>
		<tr>
			<td>Omega Inc. pressure transducer software</td>
			<td>Omega Engineering, Inc.</td>
			<td></td>
			<td>Name in Protocol: pressure transducer software</td>
		</tr>
		<tr>
			<td>Polycarbonate Disc</td>
			<td>McMaster-Carr</td>
			<td>8571K31</td>
			<td>Listed in CAD drawings for illumination port, Fig. S1 Part E</td>
		</tr>
		<tr>
			<td>Sapphire windows (x3)</td>
			<td>Guild Optical Associates, Inc.</td>
			<td>Optical Grade Sapphire Window, C-Plane 
			Diameter: 1.811&#8221; &#177;.005&#8221; 
			Thickness: .590&#8221; &#177;.005&#8221; 
			Surface Quality: 60/40 
			Edges ground and safety chamfered</td>
			<td>Buy three so there are two backups. 
			Name in Protocol: sapphire window</td>
		</tr>
		<tr>
			<td>Solid Thermocouple Wire FEP Insulation and Jacket, Type K, 24 Gauge, 50 ft. Length (x1)</td>
			<td>McMaster-Carr</td>
			<td>3870K32</td>
			<td>Name in Protocol: thermocouples</td>
		</tr>
		<tr>
			<td>Stainless Steel Integral Bonnet Needle Valve, 0.37 Cv, 1/4 in. Swagelok Tube Fitting, Regulating Stem (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-1RS4</td>
			<td>Two will be used for the pressure pump as well. 
			Name in Protocol: 1/4&#34; needle valves</td>
		</tr>
		<tr>
			<td>Stainless Steel Pipe Fitting, Hex Nipple, 1/4 in. Male NPT (x2)</td>
			<td>Swagelok</td>
			<td>&#160;SS-4-HN</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Female Branch Tee, 1/4 in. Tube OD x 1/4 in. Tube OD x 1/4 in. Female NPT (x2)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-3-4TTF</td>
			<td>Used with pressure transducer 
			Name in Protocol: branch tee fitting</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Male Connector, 1/4 in. Tube OD x 1/4 in. Male NPT (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-1-4</td>
			<td>Used on top port and side port leading to needle valves 
			Name in Protocol: NPT screws</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Port Connector, 1/4 in. Tube OD (x8)</td>
			<td>Swagelok</td>
			<td>&#160;SS-401-PC</td>
			<td>Use as tube connections between NTP and valve connections 
			Name in Protocol: port connector fitting</td>
		</tr>
		<tr>
			<td>TANK</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 OD in. x 20 ft. Copper Soft Refrigeration Coil</td>
			<td>Everbilt</td>
			<td>Model # D 04020PS</td>
			<td>For circulating coolant 
			Name in Protocol: 1/4&#34; copper pipe</td>
		</tr>
		<tr>
			<td>10 gallon aquarium</td>
			<td>Tetra</td>
			<td></td>
			<td>Name in Protocol: 10 gallon tank</td>
		</tr>
		<tr>
			<td>2 oz. Waterweld</td>
			<td>J-B Weld</td>
			<td>Model # 8277</td>
			<td>Name in Protocol: underwater sealant</td>
		</tr>
		<tr>
			<td>3 in. x 25 ft. Foil Backed Fiberglass Pipe Wrap Insulation</td>
			<td>Frost King</td>
			<td>Model # SP42X/16</td>
			<td>For wrapping around aquarium 
			Name in Protocol: foil-lined fiberglass</td>
		</tr>
		<tr>
			<td>3/8 7/8 in. Stainless Steel Hose Clamp (10 pack)</td>
			<td>Everbilt</td>
			<td>Model # 670655E</td>
			<td>Name in Protocol: worm drive hose clamps</td>
		</tr>
		<tr>
			<td>Styrofoam</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: insulating material</td>
		</tr>
		<tr>
			<td>TOOLS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-1/8 in. Ratcheting Tube Cutter</td>
			<td>Husky</td>
			<td>Model # 86-036-0111</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 in. to 1 in. Pipe Cutter</td>
			<td>Apollo</td>
			<td>Model # 69PTKC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable wrench (x2)</td>
			<td>Steel Core</td>
			<td>Model # 31899</td>
			<td>Need two wrenches with jaw at least 1&#34;</td>
		</tr>
		<tr>
			<td>Allen wrench set</td>
			<td>Home Depot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duct tape</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: duct tape</td>
		</tr>
		<tr>
			<td>Flexible tubing, like an IV line, to fit on the end of grainger probe (canula)</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: IV tube</td>
		</tr>
		<tr>
			<td>Grainger 18 gauge probe</td>
			<td>Grainger</td>
			<td></td>
			<td>For inserting droplet 
			Name in Protocol: cannula</td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow corning</td>
			<td></td>
			<td>Apply to o-rings before inserting sapphire window 
			Name in Protocol: vacuum grease</td>
		</tr>
		<tr>
			<td>Klein Tools Professional 90 Degree 4-in-1 Tube Bender</td>
			<td>Klein Tools</td>
			<td>Model # 89030</td>
			<td>Name in Protocol: tube bender</td>
		</tr>
		<tr>
			<td>Snoop liquid leak detector</td>
			<td>Swagelok</td>
			<td>MS-SNOOP-8OZ</td>
			<td>To detect leaks when pressurized when methane 
			Name in Protocol: liquid leak detector</td>
		</tr>
		<tr>
			<td>Suction cup</td>
			<td>Home Depot</td>
			<td></td>
			<td>For removing tight fitting sapphire window 
			Name in Protocol: suction cup</td>
		</tr>
		<tr>
			<td>Teflon Tape</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: plumber&#39;s tape</td>
		</tr>
		<tr>
			<td>Temflex 3/4 in. x 60 ft. 1700 Electrical Tape Black</td>
			<td>3M</td>
			<td>Model # 1700-1PK-BB40</td>
			<td>Name in Protocol: electrical tape</td>
		</tr>
	</tbody>
</table>

methane hydrate crystallization on sessile water droplets

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa working pressure, containing a stage for sessile droplets, a sapphire window for visualization, and temperature and pressure transducers. A pressure pump connected to a methane gas cylinder is used to pressurize the cell to 5 MPa. The cooling system is a 10 gallon (37.85 L) tank containing a 50% ethanol solution cooled via ethylene glycol through copper coils. This setup enables the observation of the temperature change associated with hydrate formation and dissociation during cooling and depressurization, respectively, as well as visualization and photography of the morphologic changes of the droplet. With this method, rapid hydrate shell formation was observed at ~-6 &#176;C to -9 &#176;C. During depressurization, a 0.2 &#176;C to 0.5 &#176;C temperature drop was observed at the pressure/temperature (P/T) stability curve due to exothermic hydrate dissociation, confirmed by visual observation of melting at the start of the temperature drop. The "memory effect" was observed after repressurizing to 5 MPa from 2 MPa. This experimental design allows the monitoring of pressure, temperature, and morphology of the droplet over time, making this a suitable method for testing various additives and substrates on hydrate morphology.

With this method, a gas hydrate shell on a droplet can be monitored visually through a sapphire window of the pressure cell and via temperature and pressure transducers. To nucleate the hydrate shell after pressurizing to 5 MPa, dry ice can be added to the top of the pressure cell to induce a thermal shock to trigger rapid hydrate crystallization. There is a clear morphologic difference upon dry ice-forced hydrate shell formation. The water droplet transitioned from a smooth, reflective surface (Figu...

Watch this Scientific Journal Video about Methane Hydrate Crystallization on Sessile Water Droplets at JoVE.com

Methane Hydrate Crystallization on Sessile Water Droplets

We describe a method to form gas hydrate on sessile water droplets to study the effects of various inhibitors, promoters, and substrates on the hydrate crystal morphology.

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa ...

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2.3K Views.  Georgia Institute of Technology. We describe a method to form gas hydrate on sessile water droplets to study the effects of various inhibitors, promoters, and substrates on the hydrate crystal morphology. 

Video: Methane Hydrate Crystallization on Sessile Water Droplets

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future energy resources and energy storage media. To develop a method for gas production from natural MH-bearing sediments and hydrate-based technologies, it is imperative to understand the thermal properties of gas hydrates.
The thermal properties' measurements of samples comprising sand, water, methane, and MH are difficult because the melting heat of MH may affect the measurements. To solve this problem, we performed thermal properties' measurements at supercooled conditions during MH formation. The measurement protocol, calculation method of the saturation change, and tips for thermal constants' analysis of the sample using transient plane source techniques are described here.
The effect of the formation heat of MH on measurement is very small because the gas hydrate formation rate is very slow. This measurement method can be applied to the thermal properties of the gas hydrate-water-guest gas system, which contains hydrogen, CO2, and ozone hydrates, because the characteristic low formation rate of gas hydrate is not unique to MH. The key point of this method is the low rate of phase transition of the target material. Hence, this method may be applied to other materials having low phase-transition rates.

We present a protocol for measuring the thermal properties of synthetic hydrate-bearing sediment samples comprising sand, water, methane, and methane hydrate.

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 &#176;C - 39 &#176;C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 &#176;C and 27 &#176;C), and at high temperatures (39 &#176;C) when the incubator set temperature was higher. This incubator prototype is low-cost (&lt; 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

This paper describes a method for building an adaptable, low-cost and transportable incubator for microbial testing of drinking water. Our design is based on widely available materials and can operate under a range of field conditions, while still offering the advantages of higher-end laboratory-based models.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water ...