The authors thank Miss Pi-Fen Lin, Mr. Wei-Lung Tseng, Miss Pei-Hsuan Lin, and Dr. Jia-Lu Chuan for their assistance during the field sampling and lab analysis for the practical development and application of &#34;CATNET.&#34;&#160;The assistance of crew and technician on board research vessel Ocean Research-I and Ocean Research-II during the sampling expeditions is greatly appreciated. This work was supported partly by Taiwan Ministry of Science and Technology of grants 91-2611-M-002-007, 95-2611-M-002 -009, 96-2611-M-002-004, 97-3114-M-002-006, 104-2611-M-002-019. This manuscript is written in memory of Miss Wen-Huei Lee for her immense dedication and contribution to marine researches in Taiwan.

The following protocol involves working with harmful chemicals. Please read the Safety Data Sheets (SDS) carefully, and follow institutional chemical safety guidelines.
1. Multi-layer Gravity Filtration Sampler Preparation
<ol>
	<li>Sampler cleaning
	<ol>
		<li>Fill the tubing and filtration unit with 1% (w/v) of anionic protease enzyme detergent solution and soak it for 24 h. Flush the multi-layer gravity filtration sampler with reverse osmosis double distilled water (RO-DDW) thoroughly, then fill it with 0.1% (v/v) hydrochloric acid (HCl, Reagent Grade) and soak for 72 h.</li>
		<li>Thoroughly flush the multi-layer gravity filtration sampler with reverse osmosis double distilled deionized water (RO-DD-DIW) three to five times, at least 20 liters each time, and store the assemblage in plastic bags.</li>
	</ol>
	</li>
	<li>Particle sample container cleaning/preparation
	<ol>
		<li>Use low density polyethylene (LDPE, 125 mL) or fluorinated ethylene propylene (FEP, 125 mL) bottles as containers for particles. Clean the bottles by soaking them first in alkaline detergent (Micro, 1%), then in 50% (v/v) nitric acid (HNO3, Reagent Grade), then 10% (v/v) HCl solutions for at least 24, 48, and 24 h, respectively. Rinse the bottles with de-ionized water (RO-DD-DIW) between the two soaking steps.</li>
		<li>After a final HCl soaking, rinse the bottles thoroughly with de-ionized water (RO-DD-DIW), and dry the bottles in a clean room or class-100 clean bench. 
		Attach the cleaned bottle to the multilayer gravity filtration sampler, or seal cleaned bottles in PE zippered bags and double-bag them for transport.</li>
	</ol>
	</li>
	<li>Assemblage of multi-layer gravity filtration sampler
	<ol>
		<li>Connect six 4 m long chemically resistant thermoplastic elastomer tubes (outside diameter of 0.635 cm) to the six directional inlets on top of the sampler.</li>
		<li>Assemble the three different mesh nylon filters with low-density polyethylene sample containers (125 mL LDPE) in sequence in a clean room (bench) after they are cleaned (see below), with the 10 &#181;m mesh filter positioned on the outside, the 63 &#181;m mesh filter in the middle, and the 153 &#181;m mesh filter on the inside. For transportation, store the multi-layer gravity filtration sampler in two layers of polyethylene (PE) bags, then place it in the polypropylene (PP) shipping container.</li>
	</ol>
	</li>
</ol>
2. Sampling
<ol>
	<li>Sample collection
	<ol>
		<li>Upon arrival at the sampling site, have one person remove the multi-layer gravity filtration sampler from a shipping container on the deck of the research vessel and open the bag with the sampler. Then, have them put on the PE gloves, connect the six 4 m thermoplastic elastomer tubes to the water spigots of six 20 L PTFE-coated sampling bottles on the elevated multi-bottle sampling array, and guide the seawater into this filtration unit. The seawater will flow through the directional inlets, and the particles/plankton will be gently separated/fractionated through the nets and settle into the 125 mL LDPE bottles that are secured at the base of the nets.</li>
		<li>After the seawater has flowed through (usually 120 L for coastal sea water and 240 L for open ocean water), remove each net in sequence (firstly, the 153 &#181;m, then the 63 &#181;m, and finally the 10 &#181;m) in a class-100 clean bench, then spray the net with trace-metal-clean 0.4 &#181;m filtered seawater to rinse out any plankton stuck on the inner surface of the nets. Collect the seawater with concentrated particles/plankton in 125 mL polyethylene bottles.</li>
		<li>Unscrew these bottles from the nets, and filter the solutions with concentrated particles/plankton again through an acid-washed vacuum filtration apparatus with pre-weighed, acid-washed 47 mm, 10 &#181;m pore-size polycarbonate filters under low-vacuum conditions (&#60;5 kPa).</li>
		<li>To collect particles/plankton smaller than 10 &#181;m, wait for at least 20 L of seawater to flow through the sampler, then after that, collect two to five L of water in the 5 L PE container, and filter these sample waters by an acid-washed vacuum filtration apparatus with pre-weighed, acid-washed, 47-mm, 0.4-&#181;m pore size polycarbonate filters.</li>
		<li>After vacuum filtration, rinse the sample filters immediately with high purity DDW water to remove the residue of seawater, minimizing the influence of sea salts on determining the dry weights of particles/plankton. Keep the rinsing volume to only a couple of milliliters to prevent damaging the fragile plankton.</li>
		<li>Then, after this rinsing step, carefully remove the filter from the vacuum filtration unit, store the sample filters in acid-washed, pre-weighed acrylic plastic petri dishes, and seal in resealable plastic bags. Keep the bags in a -20 &#730;C freezer onboard until returning back to a land-based laboratory for further sample pretreatment and chemical analysis.</li>
	</ol>
	</li>
</ol>
3. Sample Treatment
<ol>
	<li>Freeze drying and digestion of particles
	<ol>
		<li>Place the filters with particle samples in the collector chamber of the freeze-drying machine, and turn on the machine. As the machine temperature reaches -40 &#176;C, turn on the vacuum pump of the machine and start the freeze drying processes. 
		NOTE: The vacuum level should be maintained steadily below 0.12 mBar. Please read the user manual carefully and follow the manufacturer&#39;s guidelines for each step.</li>
		<li>After 72 h, turn off the freeze-drying machine, remove the dried filters and weigh them. Then, place dried sample filters into pre-weighed perfluoroalkoxy alkane (PFA) vessels (60 mL capacity), and add 3 mL of concentrated ultrapure nitric acid into the vessels2,3,6,7.</li>
		<li>Tighten the vessels with a torque wrench to a constant torque of 2.5 kg-m, and place the vessels in a conventional oven at 130 &#176;C for 12 h for the first digestion sequence. After cooling, remove the vessels from the oven, open the vessels, and add 2 mL of ultrapure hydrofluoric acid into the vessels2,3,6,7.</li>
		<li>Tighten the vessels with a torque of 2.5 kg-m, and place the vessels in a conventional oven at 130 &#176;C for 12 h, which is the second digestion sequence. After cooling, open the vessels and add 16 mL of 4.5% ultra-pure boric acid solution into the vessels2,3,6,7.</li>
		<li>Tighten the vessels to a constant torque of 2.5 kg-meters, and digest the samples in oven at 130 &#176;C for 12 h for the final digestion sequence. After cooling, weigh each vessel and determine the final mass and specific mass of each digested solution to yield a final digestant volume. 
		NOTE: Specific mass is determined by measuring the weight of exactly 1.00 mL of digestant.</li>
		<li>Carefully pour the digestant into 30 mL acid cleaned PE bottle for further trace metal analysis.</li>
	</ol>
	</li>
	<li>Trace Metal Analysis
	<ol>
		<li>Determine trace metal concentrations (Cd, Cu, Fe, Mn, Ni, and Al) in digested solutions of particles using a graphite furnace atomic absorption spectrometer (GF-AAS)6.</li>
		<li>As an accuracy test, use certified reference material (CRM), such as marine sediment reference materials from the National Research Council of Canada, estuarine sediment standard reference material from the National Institute of Standards and Technology of the USA, and plankton reference material from the European Commission&#39;s science and knowledge service. The process gives 95% to 107% recovery of the certified value for the trace metals provided in the CRM.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jeandel, C., 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=What+did+we+learn+about+ocean+particle+dynamics+in+the+GEOSECS-JGOFS+era.">What did we learn about ocean particle dynamics in the GEOSECS-JGOFS era.</a> Progr. Oceanogr. 133, 6-16 (2015).</li><li>Lam, 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=Methods+for+analyzing+the+concentration+and+speciation+of+major+and+trace+elements+in+marine+particles.">Methods for analyzing the concentration and speciation of major and trace elements in marine particles.</a> Progr. Oceanogr. 133, 32-42 (2015).</li><li>Collier, R., Edmond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trace+element+geochemistry+of+marine+biogenic+particulate+matter.">The trace element geochemistry of marine biogenic particulate matter.</a> Progr. Oceanogr. 13, 113-199 (1984).</li><li>Donat, J. R., Bruland, K. W., Steinnes, E., Salbu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+elements+in+the+oceans.">Trace elements in the oceans.</a> Trace Elements in Natural Waters. , 247-280 (1995).</li><li>Wen, L. -. S., Santschi, P., Tang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+radioactively+labeled+colloids+and+natural+particles:+evidence+for+colloidal+pumping.">Interaction between radioactively labeled colloids and natural particles: evidence for colloidal pumping.</a> Geochim. Cosmochim. Ac. 61, 2867-2878 (1997).</li><li>Wen, L. -. S., Warnken, K., Santschi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organic+carbon,+iron,+and+aluminium+oxyhydroxides+as+trace+metal+carriers:+Comparison+between+the+Trinity+River+and+the+Trinity+River+Estuary+(Galveston+Bay,+Texas).">The role of organic carbon, iron, and aluminium oxyhydroxides as trace metal carriers: Comparison between the Trinity River and the Trinity River Estuary (Galveston Bay, Texas).</a> Mar. Chem. 112, 20-37 (2008).</li><li>Hurd, D., Spencer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+particles:+analysis+and+characterization.">Marine particles: analysis and characterization.</a> American Geophysical Union. , (1991).</li><li>Martin, J. H., Knauer, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elemental+composition+of+plankton.">The elemental composition of plankton.</a> Geochim. Cosmochim. Ac. 37, 1639-1653 (1973).</li><li>Morel, F., Price, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biogeochemical+cycles+of+trace+metals+in+the+oceans.">The biogeochemical cycles of trace metals in the oceans.</a> Science. 300, 944-947 (2003).</li><li>Ho, T. -. Y., 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=The+elemental+composition+of+some+marine+phytoplankton.">The elemental composition of some marine phytoplankton.</a> J. Phycol. 39, 1145-1159 (2003).</li><li>McDonnell, A., 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=The+oceanographic+toolbox+for+the+collection+of+sinking+and+suspended+marine+particles.">The oceanographic toolbox for the collection of sinking and suspended marine particles.</a> Prog. Oceanogr. 133, 17-31 (2015).</li><li>Wen, L. -. S., Li, W. -. H., Zhuang, G. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Multiple layer filtering and collecting device. , (2005).</li><li>Ho, T. -. Y., Wen, L. -. S., You, C. -. F., Lee, D. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trace-metal+composition+of+size+fractionated+plankton+in+the+South+China+Sea:+biotic+versus+abiotic+sources.">The trace-metal composition of size fractionated plankton in the South China Sea: biotic versus abiotic sources.</a> Limnol. Oceanogr. 52, 1776-1788 (2007).</li><li>Hsu, R., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+estimations+of+the+density+and+porosity+of+flocs+of+varying+sizes+in+a+submarine+canyon.">In-situ estimations of the density and porosity of flocs of varying sizes in a submarine canyon.</a> Mar. Geol. 276, 105-109 (2010).</li><li>Liao, W. -. H., Yang, S. -. C., Ho, T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+metal+composition+of+size-fractionated+plankton+in+the+Western+Philippine+Sea:+the+impact+of+anthropogenic+aerosol+deposition.">Trace metal composition of size-fractionated plankton in the Western Philippine Sea: the impact of anthropogenic aerosol deposition.</a> Limnol Oceanogr. , (2017).</li><li>Grasshoff, K., Kremling, K., Ehrhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of seawater analysis. , (2007).</li></ol>

The co-author, Mr. Alan Chuang is the patent co-owner and the general manager of the company (Sino Instruments Co., Ltd.) which manufactured this collection apparatus for interested users. The patent ended in May 9th, 201512.

Obtaining reliable trace metal concentrations on plankton and suspended particles in natural waters, which are generally present at very low concentrations, requires great care during sample collection, processing, pretreatments, and analysis, with the aim of reducing contamination. Therefore, the procedures to design and prepare sampling gear, sample containers, and materials used to collect and process samples are all critical steps toward obtaining high-quality data for trace metals in marine environments. With the ad...

Particles in the ocean play an important role in marine biogeochemical cycles1. Most of the properties of particles, such as size, mineralogy, and composition, can change profoundly from one geological or hydrographical setting to another2. In addition, the distributions of elements in the ocean are also associated with the life cycle of marine phytoplankton: growth, death, sinking, and re-mineralization3,4. Marine particles span at least 4 orders of magnitude in size, ranging from submicron particles to large aggregates (&#62;5 mm). Most particles are biologically derived, from processes such as viral lysis, exudation, secretion, fecal pellet production, etc. Other particles are formed from physical coagulation of cells, cellular debris, or lithogenic materials1. Various chemical and biological characteristics of particles control both the geochemical cycles and biological processes occurring on and within the particles4,5,6. These particles are important habitats as well as food sources for some organisms, such as zooplankton or saprotrophs. Accordingly, the fate of particles is often related to their size, which can be modified by biological processes on and around particles.
Sampling marine particles usually requires filtration, but this approach introduces a certain ambiguity in identifying the properties of particles, since marine particles are not homogenous in composition and size. Suspended particles, mainly composed of small and low density particles that are almost permanently in suspension, are mixed with varying amounts of larger and denser particles in suspension only for a short period of time, depending on hydrodynamic conditions7. The first reports of the trace metal composition of plankton samples were collected by plankton tows or suspending plankton nets on a research vessel8. The authors often found metal particles and paint chips in samples, suggesting a severe problem of contamination during marine particle sampling for chemical analysis. Other efforts include net towing by rubber rafts or using a polyvinyl chloride (PVC)-hand winch3. The difficulty of reliable sampling of particles makes progress in our understanding of the chemical composition of marine particles more difficult, especially for trace elements. As such, most crucial information on the concentration of trace elements in phytoplankton has come from culture studies9,10. This recognition has motivated marine scientists to create new methods for studying particles in the sea over the past thirty years11.
Oceanographers have used various sampling techniques, including shipboard filtration, in situ filtration, and sediment traps11. The processing of large volumes of sea-water to collect non-contaminated samples can be challenging, especially for open ocean and deep waters in which the particle concentrations are very low (0.001 - 0.1 mg/L). It is also necessary to filter large volumes of sea-water to obtain an adequate quantity of particles to measure trace metal concentrations. Some researchers have used the size-fractionation method to separate suspended particles from sinking particles. However, particle size, porosity, density, and shape can all influence particle sinking velocities. Sediment traps are not practical tools to collect suspended particles, since those are designed for sinking particles. Therefore, it is important to develop sampling and treatment methods that can collect sufficient quantities of suspended particles with minimal contamination. Hence, size-fractionation by in situ filtration is still a promising tool in the oceanographer&#39;s sampling toolbox, since it can reveal critical information on marine particle dynamics. Here, we describe a successfully tested trace-metal-clean, multi-layer gravity filtration sampling apparatus, which can treat large volumes (120 - 240 L) of seawater on board in one pass from polytetrafluoroethylene (PTFE) coated water sampling bottles in a multi-bottle sampling array. This sampling apparatus uses acid-washed synthetic nylon nets in sequence, and the nets are enclosed within a polycarbonate container to gently collect size-fractionated suspended matter and phytoplankton12,13,14,15 (Figure 1). The aim of this work is to provide a better tool for studying the metal-particle associations and their reaction dynamics in marine environments, and improve our understanding of the fate of a wide variety of planktons, particles, and trace metals in these environments.

<table><tbody><tr><td>thermoplastic elastomer (C-Flex) Tubings</td><td>Cole Palmer</td><td>EW-06424-67</td><td>O.D. 0.635 cm, Opaque White 1/8&quot;ID x 1/4&quot;OD, 25 ft/pack</td></tr><tr><td>LDPE Bottle (Nalgene)</td><td>ThermoFisher Scientific</td><td>2103-0004</td><td>125 mL, Nalgene Wide-Mouth LDPE Bottles with Closure</td></tr><tr><td>anionic protease enzyme detergent detergent (Tergazyme)</td><td>Alconox</td><td>1104-1</td><td>1&amp;times;4 lb box (1.8 kg)</td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>258148</td><td>Reagent grade</td></tr><tr><td>Nitric acid</td><td>Sigma-Aldrich</td><td>695025</td><td>Reagent grade</td></tr><tr><td>alkaline detergnet (Micro)</td><td>Cole Palmer</td><td>EW-99999-14</td><td>Micro-90 Cleaning Solution</td></tr><tr><td>polycarbonate filter, 47 mm, 0.4 &amp;micro;m</td><td>Sigma-Aldrich</td><td>WHA111107</td><td>Whatman Nuclepore Track-Etched Membranes, diam. 47 mm, pore size 0.4 &amp;mu;m, polycarbonate</td></tr><tr><td>polycarbonate filter, 47 mm, 10 &amp;micro;m</td><td>Sigma-Aldrich</td><td>WHA111115</td><td>Whatman Nuclepore Track-Etched Membranes, diam. 47 mm, pore size 10 &amp;mu;m, polycarbonate</td></tr><tr><td>PFA vessel, 60 ml capacity</td><td>Savillex</td><td>300-060-03</td><td>60 mL Digestion Vessel, Flat Interior, Flat Exterior, Buttress Threaded Top</td></tr><tr><td>Nitric acid, ultrapure</td><td>Seastar Chemicals</td><td>N/A</td><td>BASELINE Nitric Acid</td></tr><tr><td>HF, ultrapure</td><td>Seastar Chemicals</td><td>N/A</td><td>BASELINE Hydrofluoric Acid</td></tr><tr><td>Boric acid, ultrapure</td><td>Seastar Chemicals</td><td>N/A</td><td>BASELINE Hydrobromic Acid</td></tr><tr><td>polyethylene (PE) gloves</td><td>Safty Zone</td><td>GDPL-MD-5</td><td>Clear Powder Free Polyethylene Gloves</td></tr><tr><td>Multiple layer filtering and collecting device</td><td>Sino Instrumnets Co. Ltd</td><td>not available</td><td>Multiple layer filtering and collecting device, CATNET</td></tr><tr><td>10 um Nylon filters, Nitex</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX 10</td><td>Nitex - Standard Widths (40 - 44 inches)</td></tr><tr><td>60 um Nylon filters, Nitex</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX 60</td><td>Nitex - Standard Widths (40 - 44 inches)</td></tr><tr><td>150 um Nylon filters, Nitex</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX 150</td><td>Nitex - Standard Widths (40 - 44 inches)</td></tr><tr><td>torque wrench</td><td>Halfords</td><td>200238</td><td>Halfords Professional Torque Wrench 8-60Nm</td></tr><tr><td>multi-bottle sampling array, Rosette</td><td>General Oceanics</td><td>Model 1018</td><td>Rosette Sampler</td></tr><tr><td>PTFE-coated sampling bottles, GO-Flo</td><td>General Oceanics</td><td>108020T</td><td>GO-Flo water sampler teflon coated</td></tr><tr><td>Marine sediment reference materials</td><td>National Research Council Canada</td><td>MESS-3</td><td></td></tr><tr><td>Estuarine sediment standard reference material</td><td>National Institute of Standards and Technology</td><td>1646a</td><td></td></tr><tr><td>Plankton reference material</td><td>The European Commission&#039;s science and knowledge service</td><td>CRM414</td><td></td></tr></tbody></table>

an ultra-clean multilayer apparatus for collecting size fractionated marine plankton and suspended particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

With the development of modern oceanography, it is now a common practice to use &#34;clean techniques&#34; to obtain accurate trace metal concentrations in marine particles or plankton. Since most particles in natural waters are in the low mg/L to &#181;g/L range, the treatment of large volumes of seawater is necessary to investigate geochemical and biological effects of trace metals on various particles in ambient environments. With the use of clean, multi-layer gravity filtration (&#34;...

Watch this Scientific Journal Video about An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles at JoVE.com

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

an-ultra-clean-multilayer-apparatus-for-collecting-size-fractionated

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

8.8K Views.  National Taiwan University. Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

Video: An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

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 ...

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Biology

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Bioengineering

Separation and Identification of Conventional Microplastics from Farmland Soils

Microplastics (MPs) pollution in the terrestrial environment has received increasing attention over the last decade, with increasing studies describing the numbers and types of MPs in different soil systems and their impacts on soil and crop health. However, different MPs extraction and analytical methods are used, limiting opportunities to compare results and generate reliable evidence for industry advice and policymakers. Here, we present a protocol that describes the methodology for sampling, separation, and chemical identification of conventional MPs&#160;from soil. The method is low-cost, and the materials are readily available. This enhances operational ease and may help with widespread adoption. The protocol provides detailed information on sample collection from the top 0-30 cm of soil using plastic-free utensils; simulation of different soil types through the use of various solid media (such as bentonite clay, silicon dioxide, and non-contaminated soil), with the addition of the same mass&#160;of polyethylene(PE)-MPs for subsequent quantification; density separation of plastic particles utilizing saturated sodium chloride (NaCl) solution and digestion of organic impurities in the supernatant using 4 M sodium hydroxide (NaOH) solution; quantification of particles using fluorescent microscopy after Nile Red staining; and polymer identification using micro&#160;Fourier-Transform Infrared Spectroscopy (&#956;-FTIR) or Laser-Direct Infrared (LDIR) spectroscopy. The MPs recovery rate ranged from 83% - 90% for the abovementioned media. This protocol presents an efficient method for soil MPs analysis that is optimized for feasibility, applicability, and cost-effectiveness. Moreover, the video accompanied can guide the process of analyzing the soil MPs step-by-step virtually. This study is dedicated to standardizing the methods for soil MPs analysis, enhancing the connectivity and comparability of measurements, and establishing a foundation for more standardized and scientific research.

Presented here is a method for extracting microplastics from soil and identifying their polymer types. The method has been optimized for execution, applicability, and cost-effectiveness. It lays a scientific foundation for standardizing the analytical method to identify microplastics in soils.

Microplastics (MPs) pollution in the terrestrial environment has received increasing attention over the last decade, with increasing studies describing ...

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

Large Volume (20L+) Filtration of Coastal Seawater Samples

Small Volume (1-3L) Filtration of Coastal Seawater Samples

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

Separation and Identification of Conventional Microplastics from Farmland Soils

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device