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[DESCRIPTION]
<p><strong>Purpose:</strong> To evaluate the influence of nutrient availability on cellular elemental composition (quotas and stoichiometry) in marine picoeukaryotes.</p>
<p><strong>Summary:</strong> Elemental quotas and ratios are assessed under nutrient replete and deplete conditions at the same time to minimize potential variation between experiments. Cells from an exponentially growing culture are concentrated by centrifugation, washed and re-suspended in a small volume of nutrient deplete media. These concentrated cells are used to inoculate triplicate replete and deplete culture flasks at a starting density corresponding to early-exponential growth. Culture growth is monitored daily. When the mean growth rate of the nutrient deplete treatment is half or less of the replete treatment (GR<em><sub>DEP</sub></em>/GR<em><sub>REP</sub></em> &lt; 0.5), samples for cell counts and elemental analyses (dissolved nutrients; particulate carbon, nitrogen, and phosphorus) are collected.</p>

[BEFORE_START]
<p><strong>Equipment and Reagents</strong>.</p>
<p> </p>
<p><em><u>Equipment</u></em><em>:</em></p>
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<p>BD Accuri flow cytometer</p>
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<p>Sorvall RC 26 Plus centrifuge</p>
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<p>Large rotor for Sorvall centrifuge</p>
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<p>Centrifuge bottles, acid-cleaned and autoclaved</p>
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<p>Balance</p>
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<p>25 mm glass filtration units (flask, funnel, &amp; support base), acid-cleaned and autoclaved</p>
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<p>vacuum pump</p>
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<p>Advantec GF-75 25mm glass fiber filters (Sterlitech Corp #GF7525MM), pre-combusted (3 hrs at 450°C)</p>
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<p>Forceps</p>
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<p>50 mL conical tubes, acid-cleaned</p>
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<p>12-well plates or pieces of pre-combusted aluminum foil</p>
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<p>Cryovials &amp; cryocanes</p>
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<p> </p>
<p><em><u>Reagents</u></em><em>:</em></p>
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<p>Nutrient replete medium</p>
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<p>Nutrient deplete medium</p>
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<p>25% EM-grade glutaraldehyde</p>
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<p>37% formaldehyde</p>
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<p><strong> Filter Combustion Procedure</strong>.</p>
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<li>Make a double thick aluminum sheet with several folds.</li>
<li>Use ethanol-cleaned forceps to arrange a single layer of 25mm Advantec GF-75 glass fiber filters (0.3 μm nominal pore size) in each fold.</li>
<li>Combust in a muffle oven for 3 hours at 450°C (plus time to warm up).</li>
<li>Open door and allow to cool overnight before removing packet.</li>
<li>Store at room temperature in a clean ziplock bag. Only open in hood.</li>
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<p> </p>
<p><strong> Acid-cleaning Procedure</strong>.</p>
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<li>After use, rinse equipment with tap water.</li>
<li>Soak in 1% Micro soap overnight (<em>Note</em>: Keep centrifuge bottles in culture room and filter units and conical tubes in main lab).</li>
<li>Rinse 6 times with tap water and 6 times with Milli-Q water, then soak in Milli-Q overnight.</li>
<li>Rinse 4 times with Milli-Q.</li>
<li>Soak in 1:10 diluted (~3.7%) trace-metal grade HCl overnight.</li>
<li>Rinse 6 times with Milli-Q water, then soak in Milli-Q overnight.</li>
<li>Rinse 2 times with Milli-Q and air dry covered with absorbent lab mats.</li>
<li>Once dry: Wrap filter unit components in foil or sterilization bags and autoclave on gravity cycle; Tightly cap conical tubes and store in plastic bag; Loosely cap centrifuge bottles and autoclave in sterilization bags on gravity cycle.</li>
</ol>

[GUIDELINES]
<p><strong>Background</strong></p>
<p> </p>
<p>Marine phytoplankton play an important role in coupling multiple nutrient cycles because they live at the interface between the abiotic and biotic components of ecosystems. The quantitative details of how multiple nutrient cycles intersect is determined by cellular elemental composition, including both the quantity and relative ratio of elements that make up a cell. The literature clearly demonstrates that phytoplankton can vary substantially in elemental composition depending on the availability of key nutrients. This variation may reflect physiological responses to ambient conditions (e.g., nutrient availability, temperature, light, etc.) and/or robust inter-specific differences in “average” elemental composition. Likewise, an organism’s ability to modify its elemental composition in response to environmental gradients (“plasticity”) varies among species. Interspecific differences in elemental composition could lead to substantial seasonal or regional differences in community structure as well as biogeochemical variables like algal biomass and export.</p>

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