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<h1 class="section-title">Laboratory Methods and Protocols</h1>
<p>I share standardized experimental protocols for C. elegans studies and microalgae research, ensuring accuracy and reproducibility. Each method includes step-by-step instructions, required reagents, and troubleshooting tips for wet lab research.</p>
<div class="protocol">
<h2>Protocol 01: Cryopreservation and Recovery Protocol for Caenorhabditis elegans</h2>
<p>Cryopreservation is an essential method for maintaining C. elegans strains over long periods. Freezing nematodes properly ensures their viability upon thawing, allowing for reliable revival and continued research.</p>
<h3>Materials Required</h3>
<ul>
<li>C. elegans cultures</li>
<li>Nematode Growth Medium (NGM) plates seeded with E. coli OP50</li>
<li>M9 buffer (Composition: NaCl, KH₂PO₄, Na₂HPO₄, H₂O)</li>
<li>Freezing solution: Soft Agar Freezing Solution</li>
<li>Cryovials (1.5–2 ml)</li>
<li>Ice bucket</li>
<li>Sterilized metal spatula</li>
<li>-80°C freezer or liquid nitrogen storage</li>
</ul>
<h3>Step-by-Step Protocol</h3>
<ol>
<li class="step"><strong>Culturing Worms:</strong> Grow C. elegans on NGM plates with E. coli OP50. Incubate at 20°C until worms reach the just-starved L1-L2 stage.</li>
<li class="step"><strong>Harvesting Worms:</strong> Wash each plate with 1.5 ml of M9 buffer to collect worms. Transfer suspensions to a 15 ml conical tube.</li>
<li class="step"><strong>Cooling and Mixing:</strong> Place the tube on ice for 15 minutes. Add an equal volume (3 ml) of the pre-warmed Soft Agar Freezing Solution.</li>
<li class="step"><strong>Aliquoting and Freezing:</strong> Transfer 1.5 ml of the mixture into labeled cryovials. Store vials in a styrofoam box at -80°C for slow cooling.</li>
</ol>
<h3>Recovery Procedure</h3>
<ol>
<li class="step"><strong>Thawing:</strong> Retrieve a cryovial from storage. Thaw rapidly at room temperature.</li>
<li class="step"><strong>Plating and Incubation:</strong> Transfer thawed suspension onto a seeded NGM plate. Incubate at 20°C for 48–72 hours.</li>
</ol>
<h3>Key Considerations</h3>
<ul>
<li>Use of Glycerol: Reduces ice crystal formation, preventing cellular damage during freezing.</li>
<li>Cooling Rate: A slow cooling rate is essential for worm viability; rapid freezing results in poor survival.</li>
<li>Optimal Worm Stage: L1-L2 stage worms have the highest post-thaw survival rate.</li>
<li>Labeling: Always label cryovials with strain information and date.</li>
</ul>
<h3>References</h3>
<ul>
<li>Protocols.io (2020). <a href="https://www.protocols.io/view/freezing-worms-bhwfj7bn">Freezing worms protocol</a>.</li>
<li>Hayden, J., et al. (2021). <a href="https://pmc.ncbi.nlm.nih.gov/articles/PMC8546488/">Cryopreservation of dehydrated Caenorhabditis elegans</a>.</li>
</ul>
</div>
<div class="protocol">
<h2>Protocol 02: Bleaching C. elegans to Obtain Eggs and L1s</h2>
<p>Bleach solution will kill worms but will not destroy eggs because they contain a protective eggshell.</p>
<h3>CAUTION:</h3>
<p>While handling bleach, wear gloves, goggles, and a lab coat.</p>
<h3>Materials</h3>
<ul>
<li>Bleach</li>
<li>Sodium hydroxide (NaOH)</li>
<li>1× M9 buffer (Dilute 10× M9 1:10 before using; sterile)</li>
<li>Adult gravid hermaphrodites</li>
<li>100-mm seeded plates for recovery</li>
</ul>
<h3>Step-by-Step Protocol</h3>
<ol>
<li class="step"><strong>Prepare Fresh Bleach/NaOH Solution:</strong> (Do not use if older than 1 week.)</li>
<li class="step"><strong>Harvest Gravid Worms:</strong> Wash gravid worms from plates with 1× M9 buffer into a 15-ml conical tube.</li>
<li class="step"><strong>Bleaching Treatment:</strong> Add 3 ml of bleach/NaOH mix and monitor adult lysis under a microscope.</li>
<li class="step"><strong>Egg Recovery:</strong> Immediately spin for no more than 1 min at 3000 rpm.</li>
</ol>
<h3>References</h3>
<ul>
<li>Porta-de-la-Riva M, et al. Basic Caenorhabditis elegans Methods: Synchronization and Observation.</li>
</ul>
</div>
<div class="protocol">
<h2>Protocol 03: Generating Males by Heat Shock</h2>
<p>Males arise spontaneously from hermaphrodite self-fertilization at about 1 in 500 eggs laid.</p>
<h3>Materials</h3>
<ul>
<li>Six or more plates with at least five L4 hermaphrodites of the desired genotype on each</li>
<li>Incubator at 30°C</li>
</ul>
<h3>Step-by-Step Protocol</h3>
<ol>
<li class="step"><strong>Incubate the plates:</strong> with the L4 hermaphrodites for 4 to 6 hours at 30°C.</li>
<li class="step"><strong>Return the plates:</strong> to 20°C. After 3 days, screen the plates for males.</li>
</ol>
<h3>References</h3>
<ul>
<li>ScienceDirect. Cryopreservation of nematodes using vitrification.</li>
</ul>
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