<h2>
	<a name="TOC-OVERVIEW"></a>OVERVIEW</h2>
<p>
	Activity C follows Activity B and takes the class out to collect samples from their field site.&nbsp; Refresh the class as necessary, or do another &lsquo;dry run&rsquo; of the procedure. Ensure that all materials are available.</p>
<h2>
	<a name="TOC-GETTING-READY"></a>GETTING READY</h2>
<h3 style="margin-left: 40px;">
	<a name="TOC-Materials"></a>Materials</h3>
<ul style="margin-left: 40px;">
</ul>
<ul style="margin-left: 40px;">
	<li>
		See the extensive list of equipment in <a href="https://sites.google.com/site/mercuryourwatershedfriends/watershed-experiences/unit-4/inverts/b-preparing-for-sampling">Activity B. Preparing for Sampling</a></li>
</ul>
<div style="margin-left: 40px;">
	&nbsp;</div>
<div>
	<h3 style="margin-left: 40px;">
		<a name="TOC-Time-Needed"></a>Time Needed</h3>
</div>
<div>
	<h2>
		<a name="TOC-ACTIVITY-PROCEDURE"></a>ACTIVITY PROCEDURE</h2>
	<p>
		&nbsp;</p>
	<h3>
		<a name="TOC-Sampling:"></a>Sampling:</h3>
	<ol>
		<li>
			Label the sample bags before you begin. Labels should be written in permanent marker right on the outside of the outer sample bag and only on the outside. Write the label in 2 places &ndash; on the front and back of the bag, or top and bottom, in case one label gets partly wiped off. Let the writing dry before you start sampling.</li>
		<li>
			The mercury instrument needs a very small amount of material. The dish that holds the samples for analysis is about the size of an average paper clip so it is not necessary to send much material in. A single beetle can be a sample. A larger amount of soil is OK but the lab technician will mix it and pull out a small piece to analyze. A few leaves and
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								<b>Students should do the whole study as planned and interpret the results, even if they realize partway through that they might have done something different. If the protocols are changed in the middle of the study, no one will know whether the results are real or whether the protocol change created some odd results.</b></div>
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			needles would be enough to analyze litter. You do need to ensure that your sampling is representative, such that it characterizes what&rsquo;s in the field. If the litter at your site is mostly spruce needles with a few maple leaves, the sample you send in should be about that same proportion.</li>
		<li>
			To collect samples, no matter what type, work in pairs. One person is in charge of the sample bag (they are called &ldquo;Dirty Hands&rdquo;) and the other will collect the samples (they are called &ldquo;Clean Hands&rdquo;). The person collecting samples (Clean Hands) puts on a pair of gloves. Once the gloves are on, Clean Hands touches nothing but the sample he/she is collecting and any pre‐cleaned supplies. Any rinsing should be done with the Poland Spring water, not any other type of water (e.g., tap water).</li>
		<li>
			Collect samples of one or more types, as appropriate for your study.</li>
	</ol>
	<p>
		&nbsp;</p>
</div>
<div>
	<h3>
		<a name="TOC-Fish-Muscle-Plugs:-Ice-Fishing"></a>Fish Muscle Plugs: Ice Fishing</h3>
	<ol>
		<li>
			On left side dorsal area of fish, clear a small area of scales with a triple‐rinsed decontaminated stainless steel scalpel.</li>
		<li>
			Clean Hands inserts the 8 millimeter biopsy punch into the fish through the scale‐free area. The punch is inserted with a slight twisting motion cutting the skin and muscle tissue. Once full depth of punch is achieved a slight bending or tilting of the punch is needed to break off the end of the sample. Remove the biopsy punch taking care to ensure the sample remains in the punch.</li>
		<li>
			Apply a generous amount of antibiotic salve to the plug area and gently return the fish to the water.</li>
		<li>
			Using a laboratory pipette bulb placed on the end of the biopsy punch, give a quick squeeze, blowing the tissue sample onto the nylon cutting board. With scalpel remove any remaining skin.</li>
		<li>
			Using forceps place muscle sample in Ziploc bag.</li>
		<li>
			Dispose of gloves and biopsy punch. Rinse scalpel and forceps three times with Poland Spring water.</li>
	</ol>
	<h3>
		<a name="TOC-Collecting-whole-fish-fish-for-tiss"></a>Collecting whole fish, fish for tissue analysis and/or stomach contents:</h3>
	<ol>
		<li>
			If you are collecting whole fish, analyzing different fish tissues or analyzing fish stomach content you will need to euthanize the fish.</li>
		<li>
			To minimize the loss of fish from the ecosystem you are sampling take NO MORE THAN 5 individual fish of any species, and/or NO MORE THAN 3 American Eel.</li>
		<li>
			To humanely kill fish:
			<ol>
				<li>
					Do not &ldquo;play&rdquo; the fish while catching it.</li>
				<li>
					Minimize the time that the fish spends out of water prior to being euthanized.</li>
				<li>
					Record the species, length and weight of your fish (if that is part of your study design). After you have recorded weight and length stun the fish with a blow to the back of the head and then decapitate the fish.</li>
				<li>
					Dissect fish using a clean nylon cutting board and rinsed stainless steel scalpel blade and handle, scissors, and forceps (dissection can occur in the classroom after the sampling event‐ store fish on ice in labeled Ziploc bags until dissection).</li>
			</ol>
		</li>
		<li>
			Set up a fish processing center by the shore, in a suitable place. Cover the working table with plastic sheeting which can be changed if necessary, so that surfaces are always kept clean.</li>
		<li>
			To fillet a fish: (If you are interested in risk to humans from fish consumption and are just sampling muscle) An initial cut should be made from the dorsal fin to the pelvic fin, just behind the opercular flap. Run the tip of the rinsed knife along the dorsal side of the fish, from the initial cut to the caudal fin. Continue making successively deeper cuts, running the knife blade as close to the neural spines and ribs as possible. After the fillet is obtained, remove the skin. Place the skin side of the fillet down on the dissecting tray, hold on to the tail portion of the fillet, and run the knife between the skin and the muscle tissue.</li>
		<li>
			Using a nitrile‐gloved hand or pre‐rinsed forceps, Clean Hands places fish into a sample bag while Dirty Hands hold the bag. Dirty Hands puts this bag into a labeled, clean outer bag. Fresh bags and gloves are to be used for each sample, and clean any tools (like forceps) should be rinsed 3 times with the Poland Spring water between samples. Don&rsquo;t forget to label!
			<ol>
				<li>
					The instrument used to determine mercury concentration only needs a small amount of sample. The students can remove a 1‐2 gram subsample from the fillet, place in clean Ziploc bag (labeled) and submit that to the lab for analysis.</li>
			</ol>
		</li>
		<li>
			For further dissection follow the &ldquo;Salmonids in the Classroom&rdquo; dissection guide.</li>
		<li>
			If examining stomach contents‐ remove stomach and store separately.</li>
		<li>
			After weighing, place each tissue of interest in a labeled Ziploc bag. Using a nitrile‐gloved hand or pre‐rinsed forceps, Clean Hands places fish into a sample bag while Dirty Hands hold the bag. Dirty Hands puts this bag into a labeled, clean outer bag. Fresh bags and gloves are to be used for each sample, and clean any tools (like forceps) should be rinsed 3 times with the Poland Spring water between samples. Don&rsquo;t forget to label!</li>
		<li>
			For shipping: Be sure you have permission to use the cooler and use extra bags or disposable plastic containers to ensure they do not mix wih any food items. It is preferred to use a cooler that is not otherwise used for food. Place all of the labeled, double sample bags into a single, unlabeled bag (just to make it easier for the lab and so the bags don&rsquo;t shift around during shipping).</li>
	</ol>
	<h3>
		<a name="TOC-Fish-collection-using-minnow-traps-"></a>Fish collection using minnow traps or eel pots</h3>
	<ol>
		<li>
			Place about &frac12; cup dried cat food or dog food in minnow trap, close trap and deploy.</li>
		<li>
			How will you be able to find your trap again? Ask students how to best mark the location.</li>
		<li>
			Remove any fish caught from the trap‐ check traps often!</li>
		<li>
			To minimize the loss of fish from the ecosystem you are sampling take NO MORE THAN 5 individual fish of any species, and/or NO MORE THAN 3 American Eel.</li>
		<li>
			To humanely kill fish:
			<ol>
				<li>
					Minimize the time that the fish spends out of water prior to being euthanized.</li>
				<li>
					Record the length and weight of your fish (if that is part of your study design). After you have recorded weight and length stun the fish with a blow to the back of the head and then decapitate the fish.</li>
				<li>
					Place fish in labeled Ziploc bag. Using a nitrile‐gloved hand or pre‐rinsed forceps, Clean Hands places fish into a sample bag while Dirty Hands hold the bag. Dirty Hands puts this bag into a labeled, clean outer bag. Fresh bags and gloves are to be used for each sample, and clean any tools (like forceps) should be rinsed 3 times with the Poland Spring water between samples. Don&rsquo;t forget to label!</li>
				<li>
					For shipping samples: Be sure you have permission to use a cooler and use extra bags or disposable plastic containers to ensure they do not mix withany food items. It is preferred to use a cooler that is not otherwise used for food. Place all of the labeled, double sample bags into a single, unlabeled bag (just to make it easier for the lab and so the bags don&rsquo;t shift around during shipping).</li>
			</ol>
		</li>
	</ol>
	<h3>
		<a name="TOC-Identification-of-stomach-contents"></a>Identification of stomach contents</h3>
	<ol>
		<li>
			In classroom: Using rinse water and forceps pull apart the stomach and rinse contents into dissecting tray.</li>
		<li>
			Separate mass and using fish field guides and insect field guides identify as many of the prey items as possible.</li>
	</ol>
	<h2>
		<a name="TOC-SUBMIT-YOUR-SAMPLES"></a>SUBMIT YOUR SAMPLES</h2>
	<p>
		Submit your samples to the lab in a cooler, on ice, by shipping them overnight or delivering them yourself. Fill out the Chain of Custody form or the lab will not have enough information to analyze your samples.</p>
	<p>
		See the &quot;<b><font size="3"><a href="https://sites.google.com/site/mercuryourwatershedfriends/watershed-experiences/unit-4/sample-submission">Guide to sample submission</a></font></b>&quot; for more details.</p>
	<h2>
		<a name="TOC-REFLECTION-ASSESSMENT-IDEAS"></a> REFLECTION/ASSESSMENT&nbsp;IDEAS</h2>
	<h4>
		<a name="TOC-Question-Set"></a>Question Set</h4>
	A student handout is available below<br />
	<ul>
		<li>
			What types of animals did you find in your invertebrate sampling or fishing campaign?</li>
		<li>
			Did different sites produce different numbers and types of animals caught?
			<ul>
				<li>
					If the answer to the above question is &quot;yes&quot;, what site was most successful in terms of animals caught?</li>
			</ul>
		</li>
		<li>
			Did certain animals prefer certain kinds of environmental conditions (deep water, shallow water, caught at the bottom or near the top of the water column)?</li>
		<li>
			Did the outside temperature have any impact on the number of animals caught?</li>
		<li>
			What types of animals are not likely to be caught in a rock bag or kick net?</li>
		<li>
			If you used more than one method to collect invertebrates, what comparisons can you make among the methods and animals caught with each?</li>
		<li>
			Would you expect to capture the same animals if you conducted this experiment several times throughout the year? How could you test your predictions experimentally?</li>
	</ul>
	<h4>
		<a name="TOC-Assessment-opportunity"></a>Assessment opportunity</h4>
	<p>
		Observe the groups as they prepare the samples to make sure that their procedures are correct. All samples should be collected according to the protocols and techniques outlined and should be properly bagged and labeled.</p>
	<h2>
		<a name="TOC-EXTENSION-IDEAS"></a>EXTENSION IDEAS</h2>
	<p>
		&nbsp;</p>
	Have the perfect extension for an activity like this? Share it with us in the comments section below.
	<p>
		&nbsp;</p>
	<h2>
		<a name="TOC-HANDOUTS"></a>HANDOUTS</h2>
	<p>
		These documents can be viewed by clicking the links below.&nbsp; Individual PDFs of each document can be downloaded from the &quot;attachments&quot; at the bottom of this web page.</p>
	<ul>
		<li>
			<a href="https://docs.google.com/viewer?a=v&amp;pid=sites&amp;srcid=ZGVmYXVsdGRvbWFpbnxtZXJjdXJ5b3Vyd2F0ZXJzaGVkZnJpZW5kc3xneDo1MzhhODU5NGIyMTBjNGEx" target="_blank">Question Sets</a></li>
	</ul>
</div>
