Name: determination of the settling rate of clay/cyanobacterial floccules
Uploaded: 2018-06-11T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Settling Rate of Clay/Cyanobacterial Floccules at JoVE.com

The authors gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (05448, 165831 and 213411).

1. Preparing Cyanobacterial Cultures
<ol>
	<li>Preparing inoculation cultures using solid media
	<ol>
		<li>Obtain axenic cyanobacterial cells from the American Type Culture Collection or Pasteur Culture Collection. For example, the unicellular, marine Synechococcus sp. PCC 7002 was obtained from the Pasteur Culture Collection, it will be referred to hereafter as Synechococcus.</li>
		<li>Maintain Synechococcus cells on plates containing solid media (A+ liquid media<sup class...

<ol>
	<li>Macquaker, H. S., Keller, M. A., Davies, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal+blooms+and+"marine+snow":+mechanisms+that+enhance+preservation+of+organic+carbon+in+ancient+fine-grained+sediments.">Algal blooms and "marine snow": mechanisms that enhance preservation of organic carbon in ancient fine-grained sediments.</a> J. Sediment. Res. 80, 934-942 (2010).</li><li>Tyson, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sedimentation+rate,+dilution,+preservation+and+total+organic+carbon:+some+results+of+a+modeling+study.">Sedimentation rate, dilution, preservation and total organic carbon: some results of a modeling study.</a> Org. Geochem. 32, 333-339 (2001).</li><li>Piper, D. Z., Calvert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+marine+biogeochemical+perspective+on+black+shale+deposition.">A marine biogeochemical perspective on black shale deposition.</a> Earth-Sci. Rev. 95, 63-96 (2009).</li><li>Sengco, M. R., Li, A. S., Tugend, K., Kulis, D., Anderson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+red-+and+brown-tide+cells+using+clay+flocculation+I.+Laboratory+culture+experiments+with+Gymnodiniumbreve+and+Aureococcus+anophagefferens.">Removal of red- and brown-tide cells using clay flocculation I. Laboratory culture experiments with Gymnodiniumbreve and Aureococcus anophagefferens.</a> Mar. Ecol. Prog. Ser. 210, 41-53 (2001).</li><li>Guenther, M., Bozelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+algae-clay+aggregation.">Factors influencing algae-clay aggregation.</a> Hydrobiologia. 523, 217-223 (2004).</li><li>Archambault, M. -. C., Grant, J., Bricelj, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+efficiency+of+the+dinoflagellate+Heterocapsa+triquetra+by+phosphatic+clay+and+implications+for+the+mitigation+of+harmful+algal+blooms.">Removal efficiency of the dinoflagellate Heterocapsa triquetra by phosphatic clay and implications for the mitigation of harmful algal blooms.</a> Mar. Ecol. Prog. Ser. 253, 97-109 (2003).</li><li>Beaulieu, S. E., Sengco, M. R., Anderson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+clay+to+control+harmful+algal+blooms:+deposition+and+resuspension+of+clay/algal+flocs.">Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs.</a> Harmful Algae. 4, 123-138 (2005).</li><li>de Magalh&#227;es, L., Noyma, N., Furtado, L., Mucci, M., van Oosterhout, F., Husza, V., Marinho, M., L&#252;rling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+coagulants+and+ballast+compounds+in+removal+of+cyanobacteria+(Microcystis)+from+water+of+the+tropical+lagoon+Jacarepagu&#225;+(Rio+de+Janeiro,+Brazil).">Efficacy of coagulants and ballast compounds in removal of cyanobacteria (Microcystis) from water of the tropical lagoon Jacarepagu&#225; (Rio de Janeiro, Brazil).</a> Estuaries and Coasts. 40, 121-133 (2017).</li><li>Li, L., Pan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+method+for+flocculating+harmful+algal+blooms+in+marine+and+fresh+waters+using+modified+sand.">A universal method for flocculating harmful algal blooms in marine and fresh waters using modified sand.</a> Environ. Sci. Tech. 47, 4555-4562 (2013).</li><li>Miranda, M., Noyma, N., Pacheco, F. S., de Magalh&#227;es, L., Pinto, E., Santos, S., Soares, M., Huszar, V., L&#252;rling, M., Marinho, 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+efficiency+of+combined+coagulant+and+ballast+to+remove+harmful+cyanobacterial+blooms+in+a+tropical+shallow+system.">The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system.</a> Harmful Algae. 65, 27-39 (2017).</li><li>Pan, G., Chen, J., Anderson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+local+sands+for+the+mitigation+of+harmful+algal+blooms.">Modified local sands for the mitigation of harmful algal blooms.</a> Harmful Algae. 10, 381-387 (2011).</li><li>Shi, W., Tan, W., Wang, L., Pan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+Microcystis+aeruginosa+using+cationic+starch+modified+soils.">Removal of Microcystis aeruginosa using cationic starch modified soils.</a> Water Research. 97, 19-25 (2016).</li><li>Du, J., Pushkarova, R. A., Smart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-SEM+study+of+aggregate+and+floc+structure+changes+during+clay+settling+and+raking+processes.">A cryo-SEM study of aggregate and floc structure changes during clay settling and raking processes.</a> Int. J. Miner. Process. 93, 66-72 (2009).</li><li>Stevens, S. E., Porter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+in+Agmenellum+quadruplicatum.">Transformation in Agmenellum quadruplicatum.</a> Proc. Natl. Acad. Sci. U. S. A. 77, 6052-6056 (1980).</li><li>Owttrim, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicases+in+cyanobacteria:+biochemical+and+molecular+approaches.">RNA helicases in cyanobacteria: biochemical and molecular approaches.</a> Methods Enzymol. 511, 385-403 (2012).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> Microbiology. 111, 1-61 (1979).</li><li>Chamot, D., Owttrim, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cold+shock-induced+RNA+helicase+gene+expression+in+the+cyanobacterium+Anabaena+sp.+strain+PCC+7120.">Regulation of cold shock-induced RNA helicase gene expression in the cyanobacterium Anabaena sp. strain PCC 7120.</a> J. Bacteriol. 182, 1251-1256 (2000).</li><li>Sutherland, B. R., Barrett, K. J., Gingras, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clay+settling+in+fresh+and+salt+water.">Clay settling in fresh and salt water.</a> Environ. Fluid Mech. 15, 147-160 (2014).</li><li>Porra, R. J., Thompson, W. A., Kriedemann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+accurate+extinction+coefficients+and+simultaneous+equations+for+assaying+chlorophylls+a+and+b+extracted+with+four+different+solvents:+verification+of+the+concentration+of+chlorophyll+standards+by+atomic+absorption+spectroscopy.">Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.</a> Biochim. Biophys. Acta. 975, 384-394 (1989).</li><li>Liu, Y. X., Alessi, D. S., Owttrim, G. W., Petrash, D. E., Mloszewska, A. M., Lalonde, S. V., Martinez, R. E., Zhou, Q. X., Konhauser, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+reactivity+of+Synechococcus+sp.+PCC+7002:+implications+for+metal+sorption+from+seawater.">Cell surface reactivity of Synechococcus sp. PCC 7002: implications for metal sorption from seawater.</a> Geochim. Cosmochim. Acta. 169, 30-44 (2015).</li><li>Playter, T., Konhauser, K., Owttrim, G., Hodgson, C., Warchola, T., Mloszewska, A. M., Sutherland, B., Bekker, A., Zonneveld, J. -. P., Pemberton, S. G., Gingras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-clay+interactions+as+a+mechanism+for+the+preservation+of+organic+matter+and+trace+metal+biosignatures+in+black+shales.">Microbe-clay interactions as a mechanism for the preservation of organic matter and trace metal biosignatures in black shales.</a> Chem Geol. 459, 75-90 (2017).</li><li>Verspagen, J. M. H., Visser, P. M., Huisman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+with+clay+causes+sedimentation+of+the+buoyant+cyanobacteria+Microcystis+spp.">Aggregation with clay causes sedimentation of the buoyant cyanobacteria Microcystis spp.</a> Aquat. Microb. Ecol. 44, 165-174 (2006).</li><li>Avnimelech, Y., Troeger, B. W., Reed, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutual+flocculation+of+algae+and+clay:+evidence+and+implications.">Mutual flocculation of algae and clay: evidence and implications.</a> Science. 216, 63-65 (1982).</li><li>Chen, L., Men, X., Ma, M., Li, P., Jiao, Q., Lu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+release+by+Aphanothece+halophytica+inhibits+cyanobacteria/clay+flocculation.">Polysaccharide release by Aphanothece halophytica inhibits cyanobacteria/clay flocculation.</a> J. Phycol. 46, 417-423 (2010).</li><li>Pan, G., Zhang, M. -. M., Chen, H., Zou, H., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+cyanobacterial+blooms+in+Taihu+Lake+using+local+soils.+I.+Equilibrium+and+kinetic+screening+on+the+flocculation+of+Microcystis+aeruginosa+using+commercially+available+clays+and+minerals.">Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of Microcystis aeruginosa using commercially available clays and minerals.</a> Environ. Poll. 141, 195-200 (2006).</li></ol>

Flocculation catalyzed by cyanobacterial cell-clay interaction has attracted a lot of interest in the fields of ecology and engineering2,3,4,5,6,7,8,9,10,11,1...

Using present environmental conditions and processes to infer past depositional mechanisms has long been an underpinning of sedimentology. While modern depositional analogues, such as the Black Sea, have been used to understand the deposition of organic-rich, fine-grained deposits, laboratory experiments have the potential to shed additional light on the origin of shale deposits. One area of inquiry in the genesis of black shales is the deposition rate and mechanism of original formation. Traditionally, it has been hypothesized that black shales formed in environments where the sedimentation rate, primary productivity, and organic matter respiration rates promote the preservation of organic matter in the sediment1,2,3. However, the role of cyanobacterial and clay flocculation has largely remained unconsidered. This mechanism of flocculation would allow for rapid deposition of organic-rich, fine-grained sediments to occur, and does not necessitate low-oxygen. Considering this premise, this protocol has two goals: 1) measure the sedimentation rate of cyanobacterial/clay floccules, and 2) visualize the sedimentation process in real time. This methodology, in addition to geochemical analysis, has been used to demonstrate that cyanobacterial/clay flocculation may in fact be an important mechanism for shale formation1. While originally intended for modelling shale deposition, this method is applicable to other disciplines such as biology and environmental remediation where the influence of clay input on bacterial metabolism and population need to be measured.
Numerous studies have been conducted to observe the flocculation of cyanobacteria and clay, for mitigating harmful algal blooms2,3,4,5,6,7,8,9,10,11,12. However, while measuring cell concentration over time, these studies have not applied cyanobacteria/clay flocculation to modelling the deposition of the rock record. As such, these studies lack a visual component, which can be critical when modelling past sedimentological processes. Additionally, the majority of studies utilize cell-counting (e.g., Pan et al.11), which can be laborious. Our method, with recent advances in measuring cyanobacterial flocculation, determines the changes in cyanobacterial cell concentration by measuring chlorophyll a (Chl a) at discrete time intervals. Pairing Chl a measurement with visual data is a new approach, which can be used to infer depositional conditions. The images generated can also be used to calculate sedimentation rate after the work from Du et al.13. The combination of visual and numerical data strengthens the reliability of the results. Furthermore, we outline additional protocols allowing for the sedimentation of dead biomass and clay to also be observed. This is important when considering past sedimentological environments, where live and dead biomass may have co-occurred. Differences in the behavior of dead biomass during the flocculation (for example, decrease in flocculation rate) would potentially have sedimentological implications.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="665">
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	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">cyanobacteria (in this study: Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803)</td>
			<td style="width:135px;">Pasteur Culture Collection</td>
			<td style="width:147px;">PCC 7002 or PCC 6803</td>
			<td style="width:167px;">used to inoculate the plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">agar</td>
			<td style="width:135px;">Thermo Scientific</td>
			<td style="width:147px;">CM0003</td>
			<td style="width:167px;">used to fill two petri dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Petri plates (standard bacteriology, 100 x 15 mm)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">82.1473.001</td>
			<td style="width:167px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 L heat resistant Erlenmeyer flask</td>
			<td style="width:135px;">Pyrex</td>
			<td style="width:147px;">4980-125</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL heat resistant Erlenmeyer flask</td>
			<td style="width:135px;">Pyrex</td>
			<td style="width:147px;">4980-250</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nichrome inoculating loop with handle</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">14-956-103</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">tinfoil</td>
			<td style="width:135px;">Reynolds Wrap Aluminum Foil</td>
			<td style="width:147px;">89079-067</td>
			<td style="width:167px;">50 cm required; used to cover foam stopper and neck of erlenmeyer flasks</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">growth media (e.g. A+)</td>
			<td style="width:135px;"></td>
			<td style="width:147px;"></td>
			<td style="width:167px;">1050 mL required; produced using composition described in tables 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bunsen Burner</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">S95941</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">plastic tubing</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">S504591</td>
			<td style="width:167px;">1 m required; used to create the bubbling apparatus</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">sponge stopper</td>
			<td style="width:135px;">Jaece Industries Inc</td>
			<td style="width:147px;">14-127-40E</td>
			<td style="width:167px;">1 required; hole made in center for pipette; used for constructin the bubbling apparatus</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">acrylic sheet&#160;</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">Optix clear acrylic sheet model # MC-102S</td>
			<td style="width:167px;">1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">clear waterproof silicone adhesive</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">Loctite clear silicone model # 908570</td>
			<td style="width:167px;">1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">camera or video recorder</td>
			<td style="width:135px;">Panasonic</td>
			<td style="width:147px;">HC-V770 HD camcorder</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tripod</td>
			<td style="width:135px;">Magnus</td>
			<td style="width:147px;">VT-300</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">black cloth</td>
			<td style="width:135px;">primomart</td>
			<td style="width:147px;">&#160;EAN 0726670162199; Part number 680254blacknappedfr</td>
			<td style="width:167px;">1 required; duvetyne light block-out cloth; approximatly 152 x 213 cm to cover tank experiment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">heat resistant serological pipet</td>
			<td style="width:135px;">corning incorporated C708510</td>
			<td style="width:147px;">13-671-101G</td>
			<td style="width:167px;">1 required; used to create the bubbling apparatus</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sample vials&#160;</td>
			<td style="width:135px;">Dynalon</td>
			<td style="width:147px;">S30467</td>
			<td style="width:167px;">at least 12 (will vary with time interval chosen)</td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">heat resistant glass pipette</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">Corning Incorporated C708510, 13-671-101G</td>
			<td style="width:167px;">1 required; used to create the bubbling apparatus; Polystyrene serological pipet would also work, but should be connected to the tubing and stopper after the rest of the apparatus is autoclaved.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">microcentrifuge</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:147px;">22 62 120-3</td>
			<td style="width:167px;">&#160;1 required;Comparable products may be used if capable of centrifuging 1.5 -2 mL microfuge tubes at 13,000 x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">vortex machine (Vortex-Genie 2)</td>
			<td style="width:135px;">Scientific Industries, Inc</td>
			<td style="width:147px;">SI-0236</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">100% methanol</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">A412-500 SDS</td>
			<td style="width:167px;">at least 12 mL (1mL per sample) required; Caution: Flammable, toxic. Wear gloves and safety glasses. Do not use or store near ignition source. Alternate sources may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cuvettes (1.6&#160; mL, polystyrene)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">67.742</td>
			<td style="width:167px;">at least 12 required</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">spectrophotometer</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">222-271600</td>
			<td style="width:167px;">1 required; Pharmacia Biotech Novaspec ll could also be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">light bulbs</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">model # 451807; internet #205477895; store SKU #1001061538</td>
			<td style="width:167px;">6-8 bulbs required to provide light for the tank experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pipette (Pipetman Classic P1000</td>
			<td style="width:135px;">Gilson</td>
			<td style="width:147px;">F123602</td>
			<td style="width:167px;">used to collect samples</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">37 % Hydrochloric acid</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:147px;">258148</td>
			<td style="width:167px;">Caution: Corrosive and toxic. Wear lab coat, safety glasses and acid-resistant gloves while using. Prepared to 4 N before use by dilution into deionized water in a chemical fumehood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foam stopper (small)</td>
			<td style="width:135px;">Canlab</td>
			<td style="width:147px;">T 1385</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Foam stopper (large)</td>
			<td style="width:135px;">Canlab</td>
			<td style="width:147px;">T 1387</td>
			<td style="width:167px;">Requires some intact stoppers and some with a single hole through the centre</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">30 &#176;C incubator/growth room with continuous illumination</td>
			<td style="width:135px;"></td>
			<td style="width:147px;"></td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">70 % Ethanol</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">BP8201500</td>
			<td style="width:167px;">30 mL&#160; required;Caution: Toxic and flammable. Wear lab coat and safety glasses</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">hydrophobic air filter (Midisart 2000, 0.2 &#181;m)</td>
			<td style="width:135px;">Sartorius</td>
			<td style="width:147px;">17805</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">clay (e.g. kaolin)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">MFCD00062311</td>
			<td style="width:167px;">at least 50 g required</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">microfuge tubes (2 mL, polypropylene)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">72.695.500</td>
			<td style="width:167px;">Comparable products may be used. At least 12 (will vary with time interval chosen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1000 &#181;L pipet tips</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">70.762</td>
			<td style="width:167px;">1 required</td>
		</tr>
	</tbody>
</table>

determination of the settling rate of clay/cyanobacterial floccules

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

When exposed to clay, cyanobacterial cells are brought out of suspension22. This is demonstrated in the representative results given here. To determine the effect of clay on cyanobacterial populations and to observe the sedimentation rates, two experiments were conducted during which Synechococcus and Synechocystis were exposed to 50 g/L kaolin clay (Table 5&#8211;6, Figure 2&#8211;3)...

Watch this Scientific Journal Video about Determination of the Settling Rate of Clay/Cyanobacterial Floccules at JoVE.com

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

This method can help answer key questions in the field of sedimentology such as how organic rich, fine grain sediments can be deposited under oxygenated conditions. The main advantage of this technique is that both quantitative results and a visual record of sedimentation are produced. To begin, prepare Cyanobacterial cultures according to the text protocol.Then rinse one 250 milliliter and two one-liter heat resistant Erlenmeyer flasks with four molar hydrochloric acid solution followed by distilled water. Fill the three flasks with liquid media. Seal the flasks with gas permeable foam stoppers and cover the necks with aluminum foil.Then label and autoclave the flasks at 121 degrees Celsius and 100 kilopascals for 25 minutes. After this, allow them to cool to room temperature. To inoculate the media, flame sterilize an inoculation loop.Then use the loop to transfer the Cyanobacterial culture into the 250 milliliter flask that contains 50 milliliters of medium. After inoculation, resterilize the lip of the 250 milliliter flask and replace the foam stopper and aluminum foil. Next, prepare the growth chamber as outlined in the text.Transfer the inoculated 50 milliliter culture to the growth chamber and incubate with a maintained temperature of 30 degrees Celsius and agitation of 150 rpm. After successful culture growth, place the 50 milliliter culture and the one liter flask containing 400 milliliters of liquid medium in a laminar flow hood. Remove the foam stoppers and foil caps.Use a Bunsen burner to sterilize the rim of each flask. Pour the culture from the 250 milliliter flask into the one liter flask containing 400 milliliters of medium. Then resterilize the neck of the one liter flask and attach the bubbling apparatus.Finally, agitate the one liter flask at 30 degrees Celsius and 150 rpm with the bubbler apparatus attached to an air pump which circulates a humidified air mixture through the solution at the rate of 300 milliliters per minute. Incubate the culture for 120 to 168 hours at 30 degrees Celsius in the growth chamber. Using a graduated cylinder, transfer additional growth medium to the 400 milliliter culture until the final volume is one liter, thus diluting the culture.Add the solution to an acrylic tank. Label two milliliter microcentrifuge tubes and place them near the acrylic tank. Then weigh out 50 grams of kaolin clay.Use a camcorder to record the experiment. Pipette one milliliter of the Cyanobacterial sample into a labeled microcentrifuge tube. Use the sample to determine the Cyanobacterial cell count by measuring the optical density at 750 nanometers.Quickly the pour clay into the tank while vigorously stirring with a stirring stick for 10 to 15 seconds. Then use a P1000 pipette to extract one milliliter of the sample for chlorophyll a determination and record this time as T0.Take additional samples at appropriate time intervals as outlined in the text. Next, use the modified procedure outlined in the text to determine the chlorophyll a concentration in each cell sample.Use a microcentrifuge to pellet the cells from each sample at room temperature for three minutes at 13, 000 times gravity. Then use a P1000 pipette to remove the excess medium. Resuspend the cell pellet with one milliliter of 100%methanol.Incubate the samples at negative 20 degrees Celsius for 24 hours. After centrifuging the samples again, use a spectrophotometer to determine the chlorophyll a concentration. This experiment illustrates the effect of clay on Cyanobacterial populations and their settling rates.Cultures of the Cyanobacteria Synechococcus that are mixed with kaolin clay are seen to have higher settling rates than the cultures containing only Cyanobacteria. After being exposed to the clay, Cyanobacterial cells settle out of suspension within 10 minutes. Cultures of the Cyanobacteria Synechocystis mixed with clay are also seen to have higher settling rates than cultures containing only Cyanobacteria.After being exposed to clay, these Cyanobacterial cells are also brought out of suspension in 10 minutes. After watching this video, you should have a good understanding of how to measure sedimentation rates of Cyanobacteria and clay mixtures which can be used to inform depositional models of black shells.

determination-of-the-settling-rate-of-claycyanobacterial-floccules

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

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

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

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

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

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.

Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&amp;#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Determination of Total Lipid and Lipid Classes in Marine Samples

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined &#39;dissolved&#39; and &#39;particulate&#39; factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...