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.

1. Calculations for Experimental Setup
<ol>
	<li>Calculate the volume of algal and/or bacterial cultures needed for controls that will be required for the entire experiment by using Equation 1: 
	<img alt="Equation 1" src="/files/ftp_upload/52455/52455equation1.1.jpg" /> 
	Where y equals the number of controls needed per day and z equals the number of days.</li>
	<li>Calculate the volume of algal and/or bacterial cultures that are needed for co-cultures for the experiment by using Equation 2: 
	<img alt="Equation 2" src="/files/ftp_upload/52455/52455equation1.2.jpg" /> 
	NOTE: It is possible to replace &#8216;co-culture&#8217; experiments with any compound screen; just adjust the final compound, solvent and algal concentrations to fit the experimental design.</li>
	<li>Use Equations 1 and 2 to calculate the final volume of early-exponential algal culture (commonly 5 days for ~104 cells/ml but this should be determined by performing an algal growth curve) required for the experiment (this volume is needed for step 2.3) using Equation 3: 
	<img alt="Equation 3" src="/files/ftp_upload/52455/52455equation1.3.jpg" /></li>
	<li>Use Equations 1 and 2 to calculate the final volume of 104 cfu/ml bacterial culture (or desired inoculation concentration) required for the experiment (this volume is needed for step 4.2) using Equation 4: 
	<img alt="Equation 4" src="/files/ftp_upload/52455/52455equation1.4.jpg" /> 
	NOTE: Use the additional 10 ml in steps 1.3 and 1.4 to account for pipetting error and any tests that are performed (e.g., WATER-PAM, flow cytometry, microscopy, etc.) on 0 d. Increase this volume if needed to fit the experimental design. 
	NOTE: For an example calculation of an 8-day experiment see Section 10. See supplemental table for media recipes.</li>
</ol>
2. Growing Algal Cells for Experimental Setup
<ol>
	<li>Isolate or obtain an actively growing axenic algal culture.</li>
	<li>Aseptically transfer 10% of the final volume of the algal culture into sterile algal medium (e.g., L1 or similar marine algal medium, see Materials and Equipment table), ensuring a 1:9 dilution of algal culture in fresh medium. Grow the diluted alga in a diurnal incubator using previously determined growth conditions for that strain (e.g., 18 &#176;C with a 16:8 hr light:dark cycle is commonly used).</li>
	<li>When the culture has reached early-exponential phase, re-culture the alga in the same sterile algal medium (e.g., L1 or similar marine algal medium), ensure the final concentration is a 1:9 dilution and that the final volume of algae is equal to the VA calculated (step 1.3). 
	NOTE: It is important to ensure these cultures are axenic. Test all algal media and algal stock bottles for contamination by plating a 20 &#956;l aliquot onto a general marine bacterial medium (e.g., Marine Broth 2216 supplemented with 1.5% agar or similar).</li>
	<li>Grow algal culture to early-exponential phase (~104 cells/ml). 
	NOTE: Every algal strain has a unique growth curve depending on culturing conditions. Early-exponential phase (~104 cells/ml) can be determined by performing an algal growth curve based on cell density (i.e. using flow cytometry or microscopy).</li>
</ol>
3. Preparing Bacterial Cells for Inoculation
<ol>
	<li>Pre-determine the bacterial concentration (cfu/ml) and optical density (OD) of the bacterium at stationary phase by doing a growth curve in the chosen bacterial medium and growth conditions (e.g., Marine Broth 2216 at 25 &#176;C and 160 rpm, or similar). Use this information to grow the cells (step 3.3) and later to dilute the cells correctly in step 3.7.</li>
	<li>Aseptically transfer an isolated and freshly grown bacterial colony from a 1.5% agar plate (e.g., Marine Broth 2216 supplemented with 1.5% agar or similar marine bacterial solid medium) into 5 ml of bacterial liquid medium (e.g., Marine Broth 2216 or similar marine bacterial medium).</li>
	<li>Grow the bacterium to stationary phase on a rolling drum or shaker (~12 - 36 hr, depending on the bacterium). Plan the experiment so that the bacterium reaches stationary phase (~108 - 109 cfu/ml) at the same time that the algal cells have reached the early-exponential phase (~104 cells/ml).</li>
	<li>Using a pipette, wash down any biofilm attached to the test tube into the medium. Pipette 1 ml of well-mixed bacterial culture into a sterile 1.5 ml microtube. Centrifuge for 1 min at 14,000 x g.</li>
	<li>Remove and dispose of the supernatant (bacterial media) without disrupting the pellet (bacterial cells). Add 1 ml of sterile algal media (e.g., L1 or similar) to the microtube (with pellet). Vortex microtube to resuspend pellet in algal media.</li>
	<li>Second wash: repeat step 3.5. 
	NOTE: It is critical to wash the bacterial cells with algal media in order to thoroughly remove all of the bacterial media, cell detritus, excreted proteins and small molecules from the cells prior to inoculating the algae with them as this could change the nutrient composition of the algal media or introduce bioactive molecules to the screen.</li>
	<li>Serially dilute the washed bacterial cells in algal media, to a final concentration that is 100-fold more concentrated than the desired final bacterial concentration (cfu/ml). Save the microtube containing cells that have been washed and diluted in algal media for step 4.2. 
	NOTE: Plan the experiment based on having a 1:1 ratio of algae to bacteria on 0 d. To do this the desired initial bacterial concentration for the experiment is 104 cfu/ml, so in step 3.7 the initial cells should be serially diluted to 106 cfu/ml.</li>
</ol>
4. Preparing Bacteria for Experimental Setup
<ol>
	<li>Prepare 4 sterile autoclaved glass conical flasks and label them: a) &#8216;algal control flask&#8217;, b) &#8216;diluted bacterial stock flask&#8217;, c) &#8216;bacterial control flask&#8217;, and d) &#8216;co-culture flask&#8217;.</li>
	<li>Dilute the bacterial suspension from step 3.7 1:99 with sterile algal media to a final volume = VB (step 1.4). Make the dilution in the flask labeled diluted bacterial stock (step 4.1). 
	NOTE: In the previous example (step 4.2), this 1:99 dilution gives a final concentration of 104 cfu/ml. Swirl flask to mix cells.</li>
	<li>Pipette Vcontrol (step 1.1) from the diluted bacterial stock and put it in the bacterial control flask.</li>
	<li>Pipette Vcontrol of sterile algal medium into the bacterial control flask (this is a 1:1 dilution). Swirl flask to mix cells and set aside for step 7.3.</li>
	<li>Pipette Vco-culture from the diluted bacterial stock flask to the co-culture flask, set flask aside for step 6.1. 
	NOTE: When doing multiple co-cultures on the same alga at once (i.e., a co-culture of two different bacterial isolates with one control) it is necessary to re-calculate the Vco-culture for each strain individually and then repeat step 4.5 for each co-culture separately. It is also necessary to increase the VA calculated in step 1.3 to include both co-cultures shown in Equation 5: 
	<img alt="Equation 5" src="/files/ftp_upload/52455/52455equation4.5.jpg" /></li>
</ol>
5. Preparing Algae for Experimental Setup
<ol>
	<li>Gently mix the early-exponential algal culture (from step 2.4) with a wide-mouth pipette tip until cells appear well mixed.</li>
	<li>Pipette Vcontrol from the algal stock bottle to the algal control flask. Gently pipette using a 10 ml pipette. Then return the algal stock bottle in the diurnal incubator.</li>
	<li>Pipette Vcontrol of sterile algal medium to the algal control flask (this is a 1:1 dilution). Swirl to mix flask and place in the diurnal incubator, until needed for step 7.4.</li>
</ol>
6. Preparing Experimental Co-culture
<ol>
	<li>Gently pipette Vco-culture from the algal stock flask into the bacterial co-culture flask from step 4.5. Return co-culture flask to the diurnal incubator until needed for step 7.5. 
	NOTE: The concentration of bacteria in the bacterial control should equal the bacterial concentration in the experimental co-culture. Similarly, the concentration of algae in the algal control should equal the concentration of algae in the experimental co-culture. By having the same initial bacterial and algal concentrations, the population density can be compared throughout the experiment.</li>
</ol>
7. Setting up Microtiter Plates
<ol>
	<li>Divide a sterile 48-well microtiter plate as per Figure 1. Label above outer wells containing either sterile diluent or non-photosynthetic samples. 
	NOTE: Randomization of the plate wells can be done for samples taken on the same day. For example, quadrant 1 will be sampled at 1 d in the experiment; the wells that should be randomized are B2 - 4 and C2 - 4. Leave the perimeter of the plate filled with sterile solution as shown in Figure 1.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52455/52455fig1highres.jpg" /> 
Figure 1. Schematic representation of sample placement in a 48-well microtiter plate. Wells are to be filled as follows: columns 1 and 6, wells A through F (<img alt="Circle 1" src="/files/ftp_upload/52455/circle1.jpg" />) are filled with 1 ml 1x PBS (or other sterile solution/media). Rows A and F, wells 2 - 8 (<img alt="Circle 2" src="/files/ftp_upload/52455/circle2.jpg" />) are filled with 1 ml bacterial control; rows B and E wells 2 - 8 (<img alt="Circle 3" src="/files/ftp_upload/52455/circle3.jpg" />) are filled with 1 ml algal control; rows C and D, wells 2 - 8 (<img alt="Circle 4" src="/files/ftp_upload/52455/circle4.jpg" />) are filled with 1 ml co-culture. The plate is divided into 4 quadrants (A2, A5, D2, and D5) these quadrants are each specific sampling days 1 - 4, this should be randomized throughout plates and labeled accordingly. Within each day we advise randomizing the algal control (<img alt="Circle 5" src="/files/ftp_upload/52455/circle5.jpg" />) and co-culture (<img alt="Circle 6" src="/files/ftp_upload/52455/circle6.jpg" />) wells using a random number generator. Label the lid over the 1x PBS and/or bacterial control wells to prevent shading of algal cultures.
<ol start="2">
	<li>Pipette 1 ml 1x phosphate buffer solution (PBS, pH 7.4) or other sterile solution in the appropriate wells as indicated in Figure 1. Perform this slowly and with care. PBS will change the ionic strength of the algal and/or co-culture medium if it splatters in other wells so handle carefully.</li>
	<li>Pipette 1 ml of bacterial control culture into wells labeled bacterial control (Figure 1). Swirl the bacterial flask before pipetting each plate to avoid bacterial settling.</li>
	<li>Pipette 1 ml of algal control culture into wells labeled algal control using a wide mouth pipette tip (Figure 1). Swirl the algal flask as regularly as the bacterial flask. If the alga tends to sink or float, swirl as regularly as is needed to maintain a visually uniform culture.</li>
	<li>Using a wide mouth pipette tip, pipette 1 ml of co-culture in the appropriate wells (Figure 1). Swirling the co-culture flask as regularly as the algal flask.</li>
	<li>Seal each plate with parafilm and place in a diurnal incubator at the desired temperature and diurnal light cycle (18 &#176;C with a 16:8 hr light:dark cycle is commonly used). Leave the plates in the diurnal incubator until ready to take PAM readings (Section 8). Ensure all plates are oriented in the same direction to allow for consistent light exposure.</li>
	<li>Take a 20 &#956;l aliquot from the PBS, algal media, algal stock and algal control and drop plate onto an appropriate non-selective medium (e.g., Marine Broth 2216 supplemented with 1.5% agar) and incubate plates at 18 - 25 &#176;C for 72 hr to test for contamination. If growth appears in any of the solutions that should not contain bacteria, then these data should not be used.</li>
	<li>Take PAM fluorometry readings using the remaining sample from the algal control and co-culture flasks for the experimental 0 d PAM fluorometry reading (see step 8.1). Any other 0 d measurements should also be performed with the remaining algal control, bacterial control and co-culture samples.</li>
</ol>
8. Taking PAM Fluorometry Readings from Stock Samples
<ol>
	<li>Zero the WATER-PAM with sterile algal media in a clean cuvette before taking readings8.</li>
	<li>Pipette 300 &#181;l from algal control or co-culture flasks into a clean cuvette containing 2.7 ml of the same algal medium used in the experiment. Mix the sample and diluent gently with a wide mouth pipette tip.</li>
	<li>Wipe-off all fingerprints from outside of cuvette with a tissue before placing the cuvette in the WATER-PAM.</li>
	<li>Place cuvette into WATER-PAM. Cover the sample with the cap and allow it to dark-adapt for 3 min. Dark adaptation times vary depending on the species of algae and must be determined for the specific alga used in the experiment. Avoid lengthy dark adaptation times (&#62;20 min) by taking WATER-PAM readings during the middle of the dark cycle of the algal diurnal incubators16,17.</li>
	<li>After dark adaption, hit F0 button. If fluorescence readings are above 3,900, dilute sample 1:1 in algal medium. Dark-adapt for an additional 3 min and take a new reading. If the F0 or Fm readings are still above 3,900, continue to dilute the sample 1:1 in algal medium until the F0 and Fm readings are below 3,900. 
	NOTE: Make sure to account for these dilutions when recording final fluorescence: for instance if the algal sample is diluted 1:9 during the initial transfer from the well to the dilution tube, then the algal fluorescence reading of 500 should be multiplied by the inverse of the dilution factor (in this case 10) and the actual fluorescence of the tube is then 5,000.</li>
	<li>After setting F0, take a saturating pulse (SAT-Pulse) reading every 90 sec by hitting the SAT button to take Fm readings. The time interval between readings may be adjusted depending on algal strain. Discard sample.</li>
	<li>Repeat steps 8.1 - 8.6 for the remaining samples.</li>
</ol>
9. Taking PAM Fluorometry Readings from Microtiter Plates
<ol>
	<li>Label 6 sterile sample tubes of &#62;3 ml volume for wells B2 - 4 for the algal control and wells C2 - 4 for the bacterial co-culture (see plate layout in Figure 1).</li>
	<li>Aliquot 2.7 ml of sterile algal medium into each tube.</li>
	<li>Place tubes in diurnal incubator and allow them to acclimate to the temperature the alga was grown at for 30 min.</li>
	<li>Before removing the microtiter plate from the incubator ensure that light penetration into the dark acclimated wells is limited by covering the plate with aluminum foil (or similar), only remove the foil while actively transferring culture from the wells to the dilution tubes (steps 9.5 - 9.7).</li>
	<li>Aseptically mix the first microtiter plate well (well B2) with a wide mouth pipette tip by slowly pipetting up and down.</li>
	<li>Obtain the dilution tubes from the incubator and aseptically transfer 300 &#181;l from well B2 (Figure 1) to its corresponding sample tube with a wide mouth pipette tip (this is a 1:9 dilution of the sample in algal medium).</li>
	<li>Repeat steps 9.5 - 9.6 for the remaining wells (B3,4 and C2 - 4).</li>
	<li>Cover sample tubes with aluminum foil and return them to the diurnal incubator until ready for WATER-PAM.</li>
	<li>Perform WATER-PAM readings as described in steps 8.1 - 8.7. Seal the microtiter plate with parafilm before returning it to the incubator.</li>
	<li>Repeat readings at planned time intervals for the duration of the experiment. The frequency and length of sampling should be planned at the beginning of the experiment.</li>
</ol>
10. Sample Experiment
The sample experiment is a 10 day co-culture of a bacterium (Phaeobacter gallaeciensis BS107) and a microalga (Emiliania huxleyi strain (CCMP3266)). It includes an algal control, bacterial control, and a bacterial-algal experimental co-culture.
<ol>
	<li>Calculate volumes of bacterial and algal stocks required (steps 1.1 - 1.4) 
	<img alt="Equation 10" src="/files/ftp_upload/52455/52455equation10.1.jpg" /></li>
	<li>Prepare the algal stock, a VA of 40 ml, by inoculating 4 ml of alga in 36 ml medium and incubated until early-exponential growth is achieved with a cell density of ~104 cells/ml (steps 2.1 - 2.4).</li>
	<li>Prepare the bacterial stock flask by diluting 400 &#956;l of the 106 cfu/ml bacteria obtained in Section 3 to a VB of 40 ml with algal media (final concentration of bacteria will be 104 cfu/ml).</li>
	<li>Transfer half (20 ml) of the bacterial stock to the bacterial control flask and add 20 ml of algal media to make the bacterial control. Transfer half (20 ml) the algal stock to the algal control flask and add 20 ml of algal media to make up the algal control (Section 5). Transfer the remaining 20 ml of bacterial stock to the co-culture flask and mix with the remaining 20 ml of algal stock to establish the co-culture (Section 6).</li>
	<li>Pipette the 1x PBS (pH 7.4), controls, and co-culture in two pre-labeled microtiter plates (Figure 1), 1 ml per well. Use 3 ml of the remaining controls and co-cultures to make 0 d measurements (cfu counts, WATER-PAM (Section 8), microscopic observation of algal cell morphology, and algal cell fixation for flow cytometry).</li>
	<li>Take WATER-PAM readings for the algal control and co-culture once a day for 10 d, and always at the same time as the 0 d measurements were taken (Section 8).</li>
</ol>
11. Other Parameters of Interest
<ol>
	<li>Determine bacterial cfu concentration: perform a serial dilution of the bacterial control and co-culture in sterile 1x PBS (or similar), then drop plate onto Marine Broth 2216 supplemented with 1.5% agar (or similar) and observe the number of cfu that grow to determine the bacterial cfu/ml for each well18.</li>
	<li>To evaluate the algal cell concentration: fix algal control and co-culture with a 0.15% final concentration of glutaraldehyde. Incubate for 10 min in the dark, then flash freeze in liquid nitrogen, and store at -80 &#176;C. Process all samples on a flow cytometer (FACS Calibur or similar) to count algal cells.</li>
	<li>Observe algal cell morphology: algal cultures and bacterial-algal co-culture using microscopy (i.e., light microscopy, epifluorescence, or similar).</li>
</ol>

<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.

Algal growth in a miniaturized format.
The miniaturization of algal cultures to a 1 ml culture volume in a microtiter plate allows for the replication within an experiment to be increased. It is important to ensure the alga is healthy throughout an experiment; perform a growth curve (Figure 2), using the microtiter plate format to assess various algal media, to ensure the nutritional requirements of the alga are met. Additionally, it may be important to optimize the diurnal cycle...

Phytoplankton physiology has traditionally been studied in meso-scale experiments ranging from 20 ml in conical flasks to 5 L in carboys1&#8211;7. This experimental scale requires subsampling for experimental monitoring, as sacrificing replicate samples for each time point creates an unmanageable experimental setup.
The ability to increase the number of independent experiments while using the same diurnal incubator space by miniaturizing the experimental volume for algal physiology experiments will reduce or eliminate the limitations of subsampling and pseudo-replication from large volumes. A microtiter plate format has been developed for algal bioassays using a 1 ml culture volume for experimentally manipulating algae in variable conditions. This small experimental volume allows for the number of replicates to be increased, increases experimental reproducibility due to a decreased variability between replicate samples and experiments, and allows true replication while maintaining experimental controls (i.e., axenic algal cultures) for 140 days (Figure 2)12.
This microtiter plate format is easily adapted for a variety of experimental questions, such as: does a bacterium have a symbiotic, neutral or pathogenic interaction with its algal host? Is the addition of a compound stimulating or toxic to an alga? These and other questions can be addressed in a rapid high-throughput manner using this new format9&#8211;11.
A 48-well microtiter culture plate allows each 1 ml well to be an independent experimental setup that is sampled at a single time-point. Various parameters can be sampled from this 1 ml volume including, but not limited to: chlorophyll fluorescence and photochemical parameters using WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry (see Materials and Equipment table)13. WATER-PAM fluorometry is a rapid and non-invasive technique that can be used to monitor experiments performed with algae13. It allows measurement of photosynthetic efficiency and PSII health from a small culture volume (150 - 300 &#956;l of culture diluted in medium to a 2 - 4 ml volume for WATER-PAM)14,15. In addition to WATER-PAM fluorometry, this setup can be used to measure a variety of other parameters including, but not limited to: microscopy to visualize the bacteria attached to algal cells and changes in the algal cell morphology; bacterial colony forming unit (cfu) counts; and flow cytometry for algal cell counts and identifying subpopulations.

<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

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

WATER-PAM fluorometry readings.
WATER-Pulse-Amplitude-Modulated (PAM) fluorometry is a quick and efficient method to determine the fluorescence (a proxy for chlorophyll content) and photosynthetic yield (PSII health) of algal cultures. The PAM WinControl software generates a spreadsheet of raw data values for (the following are the basic parameters for dark adapted algal samples):
&#160;&#160;&#160;&#160;F0 = fluorescence of dark-adapted cells
<p class="...

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.

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

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

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10.5K Views.  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.

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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

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.

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 ...

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.

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.

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

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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.

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

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has been produced. Lithium chloride (LiCl) is the preferred agent used in livestock to induce CTA because it specifically stimulates the vomit center. In addition, LiCl is commercially available, and easy to prepare and administer using a drenching gun. Nevertheless, some factors have to be considered to obtain an effective long-lasting CTA, which allows small ruminants to graze during the cropping season. A key aspect is to use animals with no previous contact with the target plant (the plant chosen to be avoided; new feed). Due to their native neophobic feeding behavior, small ruminants can easily associate the negative feedback effects with the new feed, resulting in a strong and persistent CTA. The recommended doses are 200 and 225 mg LiCl/kg body weight (BW) for goats and sheep, respectively. To induce CTA, 100 g of the target plant should be individually offered for at least 30 min, and LiCl administered thereafter if the intake is greater than 10 g. Each time the animal eats the target plant without negative consequences, the CTA becomes weaker. Consequently, to minimize the risk of target plant consumption, it is essential to have sufficient palatable ground cover available. The presence of an alternative feed (of quality and quantity) prevents the accidental consumption of the target plant. A close monitoring of the flock is recommended to remove and re-dose any animal consuming more than 4 bites or 10 g of the target plant. At the beginning of each grazing season, check the CTA status of each animal before moving them to the crop.

Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion.

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has ...

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.

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.

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.