This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.

<ol>
	<li>Scarratt, M. G., Marchetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+microbial+responses+to+iron+enrichment+in+the+Subarctic+Northeast+Pacific:+Do+microcosms+reproduce+the+in+situ+condition?.">Assessing microbial responses to iron enrichment in the Subarctic Northeast Pacific: Do microcosms reproduce the in situ condition?.</a> Deep Sea Res Part II Top. Stud. Oceanogr. 53 (20-22), 2182-2200 (2006).</li><li>Bidle, K. D., Haramaty, L., Barcelos E Ramos, J., Falkowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+activation+and+recruitment+of+metacaspases+in+the+unicellular+coccolithophore,+Emiliania+huxleyi.">Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi.</a> Proc. Natl. Acad. Sci. U. S. A. 104 (14), 6049-6054 (2007).</li><li>Moore, L. R., Goericke, R., Chisholm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+physiology+of+Synechococcus+and+Prochlorococcus:+influence+of+light+and+temperature+on+growth,+pigments,+fluorescence+and+absorptive.">Comparative physiology of Synechococcus and Prochlorococcus: influence of light and temperature on growth, pigments, fluorescence and absorptive.</a> Mar. Ecol. Prog. Ser. 116, (1995).</li><li>Iglesias-Rodriguez, M. D., Halloran, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytoplankton+calcification+in+a+high-CO2+world.">Phytoplankton calcification in a high-CO2 world.</a> Science. 320 (5874), 336-340 (2008).</li><li>Chen, M., Tang, H., Ma, H., Holland, T. C., Ng, K. Y. S., Salley, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+nutrients+on+growth+and+lipid+accumulation+in+the+green+algae+Dunaliella+tertiolecta.">Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta.</a> Bioresour. Technol. 102 (2), 1649-1655 (2011).</li><li>Lv, J. -. M., Cheng, L. -. H., Xu, X. -. H., Zhang, L., Chen, H. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+lipid+production+of+Chlorella+vulgaris+by+adjustment+of+cultivation+conditions.">Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions.</a> Bioresour. Technol. 101 (17), 6797-6804 (2010).</li><li>Geider, R., Graziano, L., McKay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+the+photosynthetic+apparatus+of+Dunaliella+tertiolecta+(Chlorophyceae)+to+nitrogen+and+phosphorus+limitation.">Responses of the photosynthetic apparatus of Dunaliella tertiolecta (Chlorophyceae) to nitrogen and phosphorus limitation.</a> Eur. J. Phycol. 33 (4), 315-332 (1998).</li><li>MacIntyre, H. L., Cullen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cultures+to+Investigate+the+Physiological+Ecology+of+Microalgae.">Using Cultures to Investigate the Physiological Ecology of Microalgae.</a> Algal Cult. Tech. , 287-326 (2005).</li><li>Blaise, C., Vasseur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal+microplate+toxicity+test.">Algal microplate toxicity test.</a> Small-scale Freshw. Toxic. Investig. Vol. 1 Toxic. Test Methods. , 137-179 (2005).</li><li>Skjelbred, B., Edvardsen, B., Andersen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measuring+growth+and+loss+rates+in+microalgal+cultures.">A high-throughput method for measuring growth and loss rates in microalgal cultures.</a> J. Appl. Phycol. 24, 1589-1599 (2012).</li><li>Nagai, T., Taya, K., Annoh, H., Ishihara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+fluorometric+microplate+algal+toxicity+assay+for+riverine+periphytic+algal+species.">Application of a fluorometric microplate algal toxicity assay for riverine periphytic algal species.</a> Ecotoxicol. Environ. Saf. 94, 37-44 (2013).</li><li>Seyedsayamdost, M. R., Case, R. J., Kolter, R., Clardy, 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+Jekyll-and-Hyde+chemistry+of+Phaeobacter+gallaeciensis.">The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis.</a> Nat. Chem. 3 (4), 331-335 (2011).</li><li>Schreiber, U., Schliwa, U., Bilger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+recording+of+photochemical+and+non-photochemical+chlorophyll+fluorescence+quenching+with+a+new+type+of+modulation+fluorometer.">Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer.</a> Photosynth. Res. 10 (1-2), 51-62 (1986).</li><li>Jones, R. J., Ward, S., Amri, A. Y., Hoegh-Guldber, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+quantum+efficiency+of+photosystem+II+of+symbiotic+dinoflagellates+of+corals+after+heat+stress,+and+of+bleached+corals+sampled+after+the+1998+Great+Barrier+Reef+mass+bleaching+event.">Changes in quantum efficiency of photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event.</a> Mar. Freshw. Res. 51 (345), 659-668 (1998).</li><li>Beer, S., Larsson, C., Poryan, O., Axelsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+rates+of+Ulva+(Chlorophyta)+measured+by+pulse+amplitude+modulated+fluorometry.">Photosynthetic rates of Ulva (Chlorophyta) measured by pulse amplitude modulated fluorometry.</a> Eur. J. Phycol. 35 (1), 69-74 (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WATER-PAM Chlorophyll Fluorometer. Instrument Description and Information for Users. , (2013).</li><li>Maxwell, K., Johnson, G. M., Heers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence--a+practical+guide.">Chlorophyll fluorescence--a practical guide.</a> J. Exp. Bot. 51 (345), 659-668 (2000).</li><li>Herigstad, B., Hamilton, M., Heersink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+optimize+the+drop+plate+method+for+enumerating+bacteria.">How to optimize the drop plate method for enumerating bacteria.</a> J. Microbiol. Methods. 44 (2), 121-129 (2001).</li><li>Kooten, O., Snel, 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+use+of+chlorophyll+fluorescence+nomenclature+in+plant+stress+physiology.">The use of chlorophyll fluorescence nomenclature in plant stress physiology.</a> Photosynth. Res. 25 (3), 147-150 (1990).</li><li>Maxwell, K., Johnson, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence--a+practical+guide.">Chlorophyll fluorescence--a practical guide.</a> J. Exp. Bot. 51 (345), 659-668 (2000).</li><li>Schreiber, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse-Amplitude-Modulation+(PAM)+Fluorometry+and+Saturation+Pulse+Method:+An+Overview.">Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview.</a> Chlorophyll a Fluoresc. A Signat. Photosynth. , 279-319 (2004).</li><li>Roh&#225;&#269;ek, K., Bart&#225;k, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+of+the+modulated+chlorophyll+fluorescence:+basic+concepts,+useful+parameters,+and+some+applications.">Technique of the modulated chlorophyll fluorescence: basic concepts, useful parameters, and some applications.</a> Photosynthetica. 37 (3), 339-363 (1999).</li><li>Da Silva, J. M., da Silva, A. B., P&#225;dua, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulated+chlorophyll+a+fluorescence:+a+tool+for+teaching+photosynthesis.">Modulated chlorophyll a fluorescence: a tool for teaching photosynthesis.</a> J. Biol. Educ. 41 (4), 178-183 (2007).</li><li>Vieira, S., Ribeiro, L., Jesus, B., Cartaxana, P., da Silva, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthesis+assessment+in+microphytobenthos+using+conventional+and+imaging+pulse+amplitude+modulation+fluorometry.">Photosynthesis assessment in microphytobenthos using conventional and imaging pulse amplitude modulation fluorometry.</a> Photochem. Photobiol. 89 (1), 97-102 (2013).</li></ol>

The authors have nothing to disclose.

La croissance des algues dans un format miniaturisé. La miniaturisation des cultures d&#39;algues à un volume de culture de 1 ml dans une plaque de microtitration permet la réplication au sein d&#39;une expérience à être augmentée. Il est important de veiller à l&#39;algue est en bonne santé tout au long d&#39;une expérience; effectuer une courbe de croissance (Figure 2), en utilisant le format de microplaques pour évaluer divers médias algues, afin d&#39;assurer le...

Phytoplancton physiologie a traditionnellement été étudiée dans des expériences à méso-échelle allant de 20 ml dans des flacons coniques à 5 L dans des bonbonnes 1-7. Cette échelle expérimentale exige le sous-échantillonnage pour la surveillance expérimentale, comme sacrifier échantillons répétés pour chaque point de temps crée un dispositif expérimental ingérable. La capacité d&#39;augmenter le nombre d&#39;expériences indépendantes tout en utilisant le même espace d&#39;incubateur diurne par miniaturiser le volume expérimental pour des expériences de physiologie algues réduiront ou élimineront les limites de sous-échantillonnage et de pseudo-réplication à partir de grands volumes. Un format de plaque de microtitrage a été développé pour des essais biologiques d&#39;algues en utilisant un volume de culture de 1 ml pour manipuler expérimentalement algues dans des conditions variables. Ce faible volume expérimental permet pour le nombre de répétitions d&#39;augmenter, augmente la reproductibilité expérimental en raison d&#39;une diminution de la variabilité entre les échantillons répétés etexpériences, et permet une véritable réplication tout en maintenant les contrôles expérimentaux (ie, les cultures d&#39;algues axéniques) pour 140 jours (Figure 2) 12. Ce format de plaque de microtitrage peut être facilement adapté pour une variété de questions expérimentales, telles que: une bactérie ne ont une interaction symbiotique, neutre ou pathogène avec son hôte algues? Est l&#39;addition d&#39;un composé stimulant ou toxiques pour une algue? Ces questions et d&#39;autres peuvent être traitées de manière à haut débit rapide en utilisant ce nouveau format 9-11. Une plaque de culture de microtitrage 48 puits permet à chaque 1 ml ainsi d&#39;être un montage expérimental indépendant qui est échantillonné à un seul point dans le temps. Différents paramètres peuvent être échantillonnés à partir de ce volume de 1 ml, y compris, mais sans s&#39;y limiter: fluorescence de la chlorophylle et les paramètres photochimiques utilisant-modulé en amplitude EAU-Pulse (EAU-PAM) fluorométrie (voir tableau Matériaux et équipement) 13. EAU-PAM fluorométrie est une technique rapide et non invasive qui peut être utilisé pour surveiller les expériences réalisées avec des algues 13. Il permet de mesurer l&#39;efficacité photosynthétique et PSII santé à partir d&#39;un petit volume de culture (150 à 300 ul de la culture dans un milieu dilué à une 2 - volume de 4 ml d&#39;eau de water-PAM) 14,15. En plus de l&#39;eau-PAM fluorométrie, cette configuration peut être utilisée pour mesurer une variété d&#39;autres paramètres, y compris, mais sans s&#39;y limiter: la microscopie à visualiser les bactéries fixées aux cellules des algues et des changements dans la morphologie des cellules d&#39;algues; formation de colonies bactériennes unitaires (ufc) chefs d&#39;accusation; et cytométrie de flux pour le nombre de cellules d&#39;algues et des sous-populations d&#39;identification.

<table border="0" cellpadding="0" cellspacing="0" width="1233">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Name of Material/ Equipment</td>
			<td style="width:328px;">Company</td>
			<td style="width:331px;">Catalog Number</td>
			<td style="width:163px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cu. ft. Diurnal Incubator (6012-1)</td>
			<td>Caron Corporate</td>
			<td>112310-6012-1-11</td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Nunc EasYFlask 25cm2, Vent/Close Cap, 7mL working volume, 200/Cs&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N156340</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell TC Plates &#8211; 48 Well</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>353078</td>
			<td><a href="http://www.bdbiosciences.com/">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P1000 Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F123602</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P10mL Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F161201</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wide Orifice Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-468</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafine Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-470</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Finntip 10mL [Vol: 1-10mL]</td>
			<td>Thermo Fisher Scientific</td>
			<td>9402151</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WATER-Pulse Amplitude Modulation (Water-ED)</td>
			<td>Heinz Walz GmbH, Effeltrich, Germany</td>
			<td>EDEE0232</td>
			<td><a href="http://www.walz.com">www.walz.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mm diameter quartz glass cuvette (WATER-K)</td>
			<td>Caron Corporate</td>
			<td></td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium&#160;Chloride&#160;(Crystalline/Certified ACS), Fisher Chemical</td>
			<td>Thermo Fisher Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Difco Marine Broth 2216</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Bacto Agar</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L1 Medium Kit, 50L</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td><a href="http://www.ncma.bigelow.org">www.ncma.bigelow.org</a></td>
		</tr>
	</tbody>
</table>

a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry

Les procédés classiques pour la manipulation expérimentale de microalgues ont utilisé de grands volumes de culture (20 ml) à 5 L, de sorte que la culture peut être sous-échantillonné pendant toute l&#39;expérience 1-7. Le sous-échantillonnage de grandes quantités peut être problématique pour plusieurs raisons: 1) il provoque la variation du volume total et l&#39;aire de surface: rapport en volume de la culture au cours de l&#39;expérience; 2) pseudo-réplication (c.-à répliquer des échantillons de la même flacon de traitement 8) est souvent employé plutôt que de véritables répétitions (ce est à dire, l&#39;échantillonnage des traitements répétés); 3) la durée de l&#39;expérience est limitée par le volume total; et 4) les cultures axéniques ou le microbiote bactérienne d&#39;habitude sont difficiles à maintenir au cours des expériences à long terme que la contamination se produit souvent au cours de sous-échantillonnage. L&#39;utilisation de plaques de microtitration permet volumes de culture 1 ml d&#39;être utilisés pour chaque répétition, avec un maximum de 48 traitements distincts dansune plaque 12,65 cm x 8,5 x 2,2, diminuant ainsi le volume expérimental et permettant la replication extensive sans aucun traitement de sous-échantillonnage. En outre, cette technique peut être modifiée pour se adapter à une variété de formats, y compris expérimentales: bactérienne-algues co-cultures, tests d&#39;algues de la physiologie et la toxine de dépistage 9-11. Puits individuels avec une algue, une bactérie et / ou co-cultures peuvent être prélevés pour de nombreuses procédures de laboratoire, y compris, mais sans s&#39;y limiter: EAU-Pulse-modulé en amplitude (EAU-PAM) fluorométrie, la microscopie, formation de colonies bactériennes unité (ufc) comtes et cytométrie de flux. La combinaison du format de plaque de microtitration et EAU-PAM fluorométrie permet de multiples mesures rapides de rendement photochimique et d&#39;autres paramètres photochimiques avec une faible variabilité entre les échantillons, une reproductibilité élevée et évite les nombreux pièges de sous-échantillonnage une bonbonne ou fiole conique au cours d&#39;une expérience .

EAU-PAM lectures de fluoro. EAU-Pulse-modulé en amplitude (PAM) fluorométrie est une méthode rapide et efficace pour déterminer la fluorescence (un indicateur de la teneur en chlorophylle) et le rendement photosynthétique (PSII de santé) des cultures d&#39;algues. Le logiciel PAM WinControl génère une feuille de calcul des valeurs de données brutes pour (voici les paramètres de base pour sombres adaptée échantillons d&#39;algues): F = 0 fluore...

Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Un essai biologique de petit volume pour évaluer bactérienne / phytoplancton co-culture avec de l&#39;eau-Pulse-modulé en amplitude (EAU-PAM) Fluorimétrie

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

a-small-volume-bioassay-to-assess-bacterialphytoplankton-co-culture

Research

JoVE Journal

Environment

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

10.4K vues.  University of Alberta. The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Vidéo: A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Une procédure de petit volume pour la concentration virale de l&#39;eau

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

Recherche

Environnement

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Une approche Cell biorapporteur entières pour évaluer transport et la biodisponibilité des contaminants organiques dans les systèmes insaturés eau

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Un poisson-alimentation Laboratoire d&#39;essais biologiques pour évaluer l&#39;activité antiprédatrices des métabolites secondaires dans les tissus d&#39;organismes marins

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

Extraction Metrics pour les systèmes de racines en trois dimensions: Volume et surface Analyse de In-sol X-ray tomodensitométrie données

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Le benthique Échange de O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; et nutriments dissous par petit noyau incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

Mesurer la capacité de vol de l’insecte Ambrosia, Platypus Quercivorus (Murayama), utilisant un Low-Cost et petit moulin de vol construit facilement

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

Un Flexible Low Cost système hydroponique, évaluer les réactions des plantes à petites molécules dans des Conditions stériles

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

DsRNA reproductible Microinjection et bioessais sur la ponte chez les moustiques et les mouches domestiques

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Composition et analyse de la Distribution des bioaérosols sous différentes Conditions environnementales

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and increasing the recovery of coalbed methane. A visualized and constant-volume gas-solid coupling system is introduced here to investigate the influence of CO2 sorption on the physical and mechanical properties of coal. Being able to keep a constant volume and monitor the sample using a camera, this system offers the potential to improve instrument accuracy and analyze fracture evolution with a fractal geometry method. This paper provides all steps to perform a uniaxial compression experiment with a briquette sample in different CO2 pressures with the gas-solid coupling test system. A briquette, cold-pressed by raw coal and sodium humate cement, is loaded in high-pressure CO2, and its surface is monitored in real-time using a camera. However, the similarity between the briquette and the raw coal still needs improvement, and a flammable gas such as methane (CH4) cannot be injected for the test. The results show that CO2 sorption leads to peak strength and elastic modulus reduction of the briquette, and the fracture evolution of the briquette in a failure state indicates fractal characteristics. The strength, elastic modulus, and fractal dimension are all correlated with CO2 pressure but not with a linear correlation. The visualized and constant-volume gas-solid coupling test system can serve as a platform for experimental research about rock mechanics considering the multifield coupling effect.

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal&#8217;s physical and mechanical properties induced by CO2 adsorption.

Une expérience de compression uniaxiale avec co2-Bearing Coal using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and ...