<p class='jove_content'>We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).</p>

<p class="jove_content">倫理声明：動物を対象とする手順は筑波大学施設内動物実験委員会によって承認されています。 </p><p class="jove_title"> EEG / EMG記録のために電極とケーブルの作製</p><ol><li>以下の手順に従って、EEG / EMGの記録電極を準備します。 <br />注記：電極は、使い捨てであり、1動物のためにのみ使用することができます。慎重に、すべてのコネクタの配線構成を計画します。正しい方向用のコネクタ上のマークを配置します。 <ol><li> 2センチのステンレス鋼線に4ピン・ヘッダの各端子を半田付けします。簡単に言えば、ピンにワイヤーの一端を保持するワイヤーピンジョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。ピンにあまりにも多くの熱を加えないように注意してください。それ以外の場合は、ピンの周りのプラスチックが溶けてしまいます。 </li><li>先頭にピンヘッダに接続されている2線のそれぞれの自由端をはんだ付けし1.0 mmの直径のステンレス鋼製ねじの。簡単に言えば、ねじ頭部下のスレッドに、ワイヤの自由端を保持するワイヤースクリュージョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。ネジなしの2線が筋電図記録電極として機能する一方、ネジで2ワイヤは、脳波記録電極として機能します。 </li><li> EMG信号の品質を高めるために、EMG電極の端で絶縁の1ミリメートルを取り除くためにハサミを使用してください。 </li><li>完全EEG / EMGの録音中に電気的なノイズを低減するために細い木の棒や歯のピックを使用してエポキシ系接着剤ですべてのはんだ付けのピンをカバーしています。 </li></ol></li><li>以下に説明するようにスリップリングに電極を接続するためのケーブルを用意します。このケーブルを再利用することができます。 <ol><li> 30 cmのフラットケーブルの線で4ピンFFC / FPCコネクタの各ピンを半田付けします。簡単に言えば、ホットはんだごてを配置し、ピンに電線の被覆をむいた部分を保持ワイヤーピンジョイントの上に、とだけで十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。 <br />注：EEG / EMG記録のために使用される実験動物用ケージの高さに適したフラットケーブルの長さを選択してください。 </li><li>はんだは、フラットケーブルのもう一方の端のワイヤーの先端にソケットを圧着します。簡単に説明すると、ワイヤの被覆の自由端に圧着ソケットを保持するワイヤーソケットジョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。 </li><li> 4位圧着ハウジングにそれぞれ圧着ソケットを挿入します。 </li><li>完全に細い木の棒や歯のピックを使用してエポキシ系接着剤で圧着ソケットをカバーしています。 </li></ol></li></ol><p class="jove_title">マウスヘッドの電極の2注入（所要時間：約20分） </p><ol><li>手術前にホットビーズ滅菌器内のすべての手術器具を滅菌します。男性のマウスを麻酔（10から20週齢、20〜30グラム）ペントバルビタール（50mg / kg）の腹腔内注射です。マウスが深くつま先をつまんで麻酔されていることを確認した後、バリカンで頭と首の毛を剃ります。 </li><li>定位フレームにマウスを移動し、2耳のバーの間の頭を固定します。乾燥を防ぐために、目にワセリンを適用します。アルコールで剃られ、肌を清潔にし、頭蓋骨を露出するようにメスで正中線に沿ってそれをカット。オープン手術領域を維持するために皮膚をクリップ。 </li><li>超硬カッター（ドリルサイズ：0.8 mmの直径）を使用して、頭蓋骨の中に、ドリル2穴を、前頭皮質領域（正中線に横1.5ミリメートルをブレグマの1mm前方）上の1つと頭頂領域の上に他の（ 1ミリメートルラムダの前方、正中線に横1.5ミリメートル）右半球の、Paxinosとフランクリンの定位座標に応じた<sup>。23</sup> </li><li>宝石商のドライバーを使用することで、穴にステンレス製脳波記録ネジを配置し、2作る - 各SCRE 2.5ターン皮質の上に硬膜外位置決め用ワット<br />注意：脳組織への損傷を防止するにはあまりにも深いネジを挿入しないでください。ネジがしっかりと頭蓋骨に固定されていることを確認してください。これは、複数のレコーディング（典型的には、1ヶ月以上）の長い期間の間に安定したEEG信号を有することが重要です。ウィグリーネジは脳波アーティファクトを生成し、実験スケジュールの終了前にオフに来るかもしれません。 </li><li>電極アセンブリ修正歯科用セメントで頭蓋骨とカバーに瞬間接着剤で（参照：セクション1.1を、ピンが上向き）。僧帽筋（首）筋肉のピンセットで二国間の小さな穴を作り、穴にEMG電極として機能するステンレス鋼線を挿入します。筋肉の露出を避けるために絹糸（直径0.1mm）で皮膚を縫合。 </li><li>定位フレームからマウスを外します。細菌感染を避けるために、マウスを、アンピシリン（100mg / kg）および腹腔内メロキシカム（1ミリグラム/キログラム）を管理し、それぞれ、手術後の痛みを軽減します。柯熱パッドの上にマウスをEPと、それは胸骨横臥位を維持するのに十分な意識を取り戻したまでそれを監視します。ハウスは、個別に回復中のマウスは、他の動物によって電極の除去を回避し、手術後の最初の日に腹腔内、メロキシカム（1ミリグラム/キログラム）を管理します。 </li></ol><p class="jove_title"> 3.記録と脳波/ EMGデータの取得</p><ol><li> 1週間の回復期間、住宅防音記録チャンバーに入れた実験ケージに個別に各マウスの後。 12時間の光/ 12時間暗のサイクルを制御し、自動的に23±1℃の周囲温度を維持し、（照度〜100ルクス、08:00に点灯）。 </li><li>記録ケーブルにマウスの頭の上にEEG / EMG電極アセンブリを接続します。記録ケーブルが（マウスの動きがケーブルのねじれによって制限されないように設計された）スリップリングおよびEEG / EMG信号増幅器に接続されていることを確認。フィルタEEG / EMG信号（脳波、0.5-64ヘルツ; EMG、16-64 Hz）は、アナログ-デジタル変換器（A / D）で128ヘルツのサンプリングレートでデジタル化し、最終的にはEEG / EMG記録ソフトウェアを実行するコンピュータ（表「材料」、4 <sup>番目のエントリ</sup>に記録下から）。 </li><li>記録チャンバー内の3日間 -  2のためのマウスを慣らします。 EEG / EMG記録を腹腔内薬物投与が含まれる場合は、ゆっくり薬物投与時に各慣れ日にマウスを処理します。 </li><li>その後、EEG / EMG記録ソフトウェア（表「マテリアル」、下から4 <sup>番目のエントリ</sup> ）を起動します。 <ol><li> 「データファイル情報]タブを選択し、ファイル名の横にあるボックスをクリックします。ファイル名を入力し、[保存]をクリックします。 </li><li> 「記録条件」タブを選択し、記録されるすべてのEEG / EMGチャンネルを選択します。 </li><li> 「記録条件」タブで、サンプリング周波数（128 Hz）を選択します。 </li><li>選択したチャンネルが目に正しく表示されているかどうかを確認してくださいE &#39;チャンネル情報」タブをクリックします。 </li><li> 「タイマー設定」タブを選択し、脳波と筋電図を表示するには、「モニター」をクリックします。 </li><li> EEG / EMG信号が正しく表示されるか確認してください。 </li><li> 「タイマー設定」タブを選択し、「メインタイマー」領域での記録の開始と終了のクロック時刻を設定します。 </li><li>録音を開始するには「タイマー設定」タブの「モニター」をクリックします。 </li></ol></li><li>ベースラインの下に録音EEG / EMG信号（ <em>すなわち</em> 、自由に振る舞うマウスの睡眠/覚醒行動）と異なる処理条件（ <em>例えば</em> 、カフェインの投与または偽の治療）、数日間。最後の実験日後にペントバルビタール（200mgの/ kg）の腹腔内注射でマウスを安楽死させます。 </li></ol><p class="jove_title"> EEG / EMGデータに基づく行動状態の4得点</p><ol><li> EEG / EMG解析のためのソフトウェアを起動します（表「マテリアル」、下から4 <sup>番目のエントリ</sup> ）。手順3で製造したEEG / EMG生データ（.kcdファイル）を開き「スリープ」タブをクリックし、エポックの時間を選択します。 10秒を選択します。 </li><li> 「スリープ」タブをクリックし、自動的に3段階にすべての10秒エポックを採点する「マルチスクリーニング」を選択（ <em>すなわち</em> 、NREMおよびREM睡眠を、および覚醒）脳波と筋電図の振幅の基礎とパワースペクトル解析に脳波の<sup>。3</sup> </li><li> 「脳波のためのFFT条件]をクリックします。 </li><li>パワースペクトル分析のためのパラメータを設定し、[256基準点（EEGの2秒に相当）、ハニング窓関数、及びエポック当たり5スペクトルの平均<sup>]。3「OK」</sup>をクリックします。 </li><li>自動スクリーニングを開始する「スクリーニング開始]をクリックします。得点データ（.rafファイル）を開きます。 </li><li>代表的な結果、 <strong>図1B</strong> <strong>および</strong>表<strong>1を参照してください</strong> （自動スクリーニングの結果を確認し、必要に応じて、標準的な基準に基づいて手動でそれらを修正</s<sup>trong&gt;）。2、3</sup>簡単に説明すると、クリックして、間違って得点エポック上でマウスの左ボタンを押したまま、誤っ得点エポックの文字列全体にカーソルをドラッグします。マウスの左ボタンを離すと、ポップアップウィンドウに正しい行動の状態を選択します。 <br />注：時々、2警戒状態間の遷移でのエポックは、一つの状態に明確に得点することは困難です。このような場合には、エポックは、表向きの状態にスコア付けされるべきであり、同じ基準は、データの再現性を保証するために、実験全体を通して同様の時期に適用されるべきです。 </li></ol>

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<li>Huang, Z. L., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenosine+A2A%2C+but+not+A1%2C+receptors+mediate+the+arousal+effect+of+caffeine%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Adenosine A2A, but not A1, receptors mediate the arousal effect of caffeine.</a> <em>Nat. Neurosci</em>. <strong>8</strong> (7), 858-859 (2005).</li>
<li>Qu, W. M., Huang, Z. L., Xu, X. H., Matsumoto, N., Urade, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopaminergic+D1+and+D2+receptors+are+essential+for+the+arousal+effect+of+modafinil%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Dopaminergic D1 and D2 receptors are essential for the arousal effect of modafinil.</a> <em>J. Neurosci</em>. <strong>28</strong> (34), 8462-8469 (2008).</li>
<li>Huang, Z. L., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arousal+effect+of+orexin+A+depends+on+activation+of+the+histaminergic+system%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Arousal effect of orexin A depends on activation of the histaminergic system.</a> <em>Proc. Natl. Acad. Sci. USA</em>. <strong>98</strong> (17), 9965-9970 (2001).</li>
<li>Xu, Q., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+mimicking+human+first+night+effect+for+the+evaluation+of+hypnotics%5BTitle%5D)+AND+%22Pharmacol.+Biochem.+Behav%22%5BJournal%5D)">A mouse model mimicking human first night effect for the evaluation of hypnotics.</a> <em>Pharmacol. Biochem. Behav</em>. <strong>116</strong>, 129-136 (2014).</li>
<li>Cho, S., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marine+polyphenol+phlorotannins+promote+non-rapid+eye+movement+sleep+in+mice+via+the+benzodiazepine+site+of+the+GABAA+receptor%5BTitle%5D)+AND+%22Psychopharmacol%22%5BJournal%5D)">Marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the GABAA receptor.</a> <em>Psychopharmacol</em>. <strong>231</strong> (14), 2825-2837 (2014).</li>
<li>Liu, Y. Y., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Piromelatine+exerts+antinociceptive+effect+via+melatonin%2C+opioid%2C+and+5HT1A+receptors+and+hypnotic+effect+via+melatonin+receptors+in+a+mouse+model+of+neuropathic+pain%5BTitle%5D)+AND+%22Psychopharmacol%22%5BJournal%5D)">Piromelatine exerts antinociceptive effect via melatonin, opioid, and 5HT1A receptors and hypnotic effect via melatonin receptors in a mouse model of neuropathic pain.</a> <em>Psychopharmacol</em>. <strong>231</strong> (20), 3973-3985 (2014).</li>
<li>Qu, W. M., <em>et al.</em> <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipocalin-type+prostaglandin+D+synthase+produces+prostaglandin+D2+involved+in+regulation+of+physiological+sleep%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep.</a> <em>Proc. Natl. Acad. Sci. USA</em>. <strong>103</strong> (47), 17949-17954 (2006).</li>
</ol>

<p class='jove_content'>The authors Yujiro Tauguchi and Sayaka Kohtoh are employees of Kissei Comtech Co., Ltd that develops SleepSign software for acquisition and automatic scoring of EEG/EMG data used in this article.</p>

<p class="jove_content">このプロトコルは、低ノイズ、費用対効果の高い、高スループットの条件で睡眠と覚醒の評価を可能にするEEG / EMGの記録のためのセットアップについて説明します。 EEG / EMG電極ヘッドアセンブリのサイズが小さいために、このシステムは、マウスへの薬物の微量注入、光遺伝学（光ファイバの注入）または同時カニューレ注入と組み合わせて含むイントラ脳実験のための他のインプラントと...

<p class="jove_content">技術の進歩は、多くの場合、神経生物学的プロセスの理解の飛躍を沈殿させています。例えば、ヒト頭皮から記録された電位は、正弦波の形をとった、の周波数は、被験者の覚醒のレベルに直接関連していたことを1929年にハンス・ベルガーの発見は、睡眠 - 覚醒の理解の急速な進歩につながりました同様に動物およびヒトの両方で規制<sup>、。1</sup>この日にelectroencephlogram（EEG）、筋電図（EMG）と組み合わせて、 <em>すなわち</em> 。骨格筋によって生成され、電気的活動は、ほぼすべての実験的および臨床のデータ「バックボーン」を表しヒトを含む、動物を振る舞うにおける皮質ニューロンの活動と行動や生理機能を相関しようとする評価。最も基本的な睡眠研究室では、これらのEEG記録は、Dを取得し、前記ケーブルベースのシステム（ <strong>図1）を用いて</strong>実行されますATAは、[ <em>例えば</em> 、高速フーリエ変換を適用（FFT）アルゴリズム]パターンとスペクトル解析にオフラインで供される記録される対象の警戒状態を決定する<sup>。2、3</sup>睡眠は急速眼球運動（REM）で構成され、ノンレム（NREM）睡眠。 REM睡眠は、急速な低電圧EEG、ランダムな眼球運動、および筋無緊張、筋肉を効果的に麻痺している状態によって特徴付けられます。身体は、脳の大部分は切断され、深い眠りであるように見えるのに対し、脳活動は、覚醒のに似ているので、REM睡眠はまた、逆説睡眠としても知られています。対照的に、運動ニューロンは、NREM睡眠中に刺激されるが、何の眼球運動は存在しません。脳波の4 Hzの - 人間ノンレム睡眠は、ステージ4は深い眠りまたは徐波睡眠と呼ばれ、0.5の間のデルタ活性を有する大規模な、遅い脳波によって識別される、4段階に分けることができます。一方、ラットのAのような小さい動物でのノンレム睡眠の相間の細分化、NDマウスは、ヒトで見られるように、彼らは睡眠の長い連結期間を持っていない主な理由は、確立されていません。 </p><p class="jove_content">長年の間、及びEEGの解釈に基づいて、睡眠 - 覚醒の調節のいくつかのモデルは、circuit-および体液性系の両方が提案されています。神経と睡眠の必要性の細胞の基礎あるいは、「スリープ・ドライブは、「未解決のままであるが、覚醒期間中に構築し、睡眠によって放散される恒常的圧力として概念化されています。一説には、内因性somnogenic要因は覚醒時に、それらの緩やかな蓄積が睡眠恒常的圧力の基盤であることを蓄積することです。液性因子によって調節されて眠る最初の正式な仮説が1892年に発表された<sup>4</sup>ローゼンバウムの仕事に入金されているが、それは石森<sup>5、6、</sup>独立ピエロン<sup>7</sup>で、100年以上前、睡眠促進化学物質の存在を実証しました。両研究者は催眠薬物質または「hypnotoxinsは「睡眠不足の犬の脳脊髄液（CSF）中に存在したことを、提案し、実際に証明した<sup>。8</sup>過去一世紀にわたり睡眠恒常性のプロセスに関与し、いくつかの追加の推定催眠薬の物質が同定されています（レビューについて、参考文献を参照してください。9）、プロスタグランジン（PG）は、D <sub><sup>2、10</sup></sub>サイトカイン<sup>、11</sup>アデノシン<sup>、12</sup>アナンダミド<sup>、13</sup>およびウロテンシンIIペプチドを含む<sup>。14</sup> </p><p class="jove_content">初期および中期20 <sup>世紀に</sup>エコノモ<sup>15、16、Moruzzi</sup>とマーグン<sup>17、</sup>およびその他による実験研究は、睡眠と覚醒の回線ベースの理論に影響を与えた結果を生成し、ある程度までの当時の体液理論を影睡眠。今日までに、いくつかの「回路モデル」は、それぞれ、（総説については、参考文献18を参照）の品質と量を変化させるデータによって通知、提案されています。一つのモデル例えば、徐波睡眠が前脳基底部におけるコリン作動性ニューロンからのアセチルコリン放出、主にブロカの対角バンドと物質のinominataの水平手足の核のconsisitingエリアのアデノシン媒介阻害を介して生成することが提案されています。 <sup>19</sup>睡眠/覚醒調節のもう一つの人気モデルは視床下部および脳幹における腹外側視索前野における睡眠導入ニューロンおよびウェイク誘導ニューロン間の相互抑制性の相互作用に基づいて、フリップフロップスイッチ機構について説明しています<sup>。18、20、21</sup>また、レム睡眠の中と外のスイッチングのために、同様の相互に抑制相互作用は脳幹内の領域のために提案されている、それは腹側中脳水道周囲灰白質、横橋被蓋、およびsublaterodorsal核である<sup>。22</sup>まとめると、これらのモデルは貴重な証明されていますヒューリスティックと睡眠研究の研究のために与えられた重要な解釈の枠組み。しかし、あなたがた睡眠 - 覚醒周期を調節する分子機構や回路のトンの完全な理解は、そのコンポーネントのより完全な知識が必要になります。以下に詳細ポリグラフ記録するためのシステムは、この目標を支援する必要があります。 </p>

<table><tbody><tr><td>4-pin header</td><td>Hirose</td><td>A3B-4PA-2DSA(71)</td><td></td></tr><tr><td>Ampicillin</td><td>Meiji Seika</td><td></td><td></td></tr><tr><td>Analog-to-digital converter</td><td>Contec</td><td>AD16-16U(PCIEV)</td><td></td></tr><tr><td>Caffeine</td><td>Sigma</td><td>C0750</td><td></td></tr><tr><td>Carbide cutter</td><td>Minitor</td><td>B1055</td><td></td></tr><tr><td>Crimp housing</td><td>Hirose</td><td>DF11-4DS-2C</td><td></td></tr><tr><td>Crimp socket</td><td>Hirose</td><td>DF11-30SC</td><td></td></tr><tr><td>Dental cement (Toughron Rebase)</td><td>Miki Chemical Product</td><td></td><td></td></tr><tr><td>Epoxy adhesive</td><td>Konishi</td><td>#16351</td><td></td></tr><tr><td>FFC/FPC connector</td><td>Honda Tsushin Kogyo</td><td>FFC-10BMEP1(B)</td><td></td></tr><tr><td>Flat cable</td><td>Hitachi Cable</td><td>20528-ST LF</td><td></td></tr><tr><td>Instant glue (Aron Alpha A)</td><td>Toagosei</td><td>N/A</td><td></td></tr><tr><td>Meloxicam</td><td>Boehringer Ingelheim</td><td>N/A</td><td></td></tr><tr><td>Pentobarbital</td><td>Kyoritsu Seiyaku</td><td>N/A</td><td></td></tr><tr><td>Signal amplifier</td><td>Biotex</td><td>N/A</td><td></td></tr><tr><td>Sleep recording chamber</td><td>APL</td><td>N/A</td><td></td></tr><tr><td>SleepSign software</td><td>Kissei Comtec</td><td>N/A</td><td>for EEG/EMG recording/analysis</td></tr><tr><td>Slip ring</td><td>Biotex</td><td>N/A</td><td></td></tr><tr><td>Stainless steel screw</td><td>Yamazaki</td><td>N/A</td><td>&amp;phi;1.0 &amp;times; 2.0</td></tr><tr><td>Stainless steel wire</td><td>Cooner Wire</td><td>AS633</td><td></td></tr></tbody></table>

polygraphic recording procedure for measuring sleep in mice

<p class='jove_content'>Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity. </p>

<p class="jove_content" fo:keep-together.within-page="1"> <strong>図1Bは、</strong>異なる警戒状態でマウスの脳波の例を示します。 <strong>表1</strong>に示すように、脳波は4 Hz以下デルタリズムで大規模な、遅い脳波を示し、EMGが弱いまたは無信号を持っている場合、エポックは、ノンレム睡眠に分類されます。脳波は、6と10 Hzの間のシータ範囲で急激な低電圧脳波を示し、EMGは低振幅を示している場合エポックは、レム睡眠に分...

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

<p class='jove_content'>The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.</p>

マウスで睡眠を測定するためのポリグラフ記録手順

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

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23.5K ビュー.  University of Tsukuba. The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

ビデオ: Polygraphic Recording Procedure for Measuring Sleep in Mice

Electrolytic Cells

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Electrolytic Cells<button class="expand">Expand</button>
	<p>In this experiment, you will be assembling an electrolytic cell to perform electroplating. An electrolytic cell contains an anode and cathode connected by a power source, which drives the nonspontaneous redox reaction in the cell. When the anode material is oxidized, the ions travel to the cathode, where they are reduced and plated.</p>
	<p>In the case of this experiment, the electrolytic cell contains a copper anode and a brass key for the cathode. Oxidation of the anode creates copper(2+) ions, which are then reduced on the brass key, forming a thin plating of solid copper.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on gloves, safety goggles, and a lab apron, which must be worn at all times.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Obtain one 10-ohm resistor, one copper sheet electrode, one copper wire, and one piece of emery paper.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use the emery paper to polish the brass key and the copper sheet. Rinse the key and the copper electrode with water. The brass key will be the cathode, and the copper sheet will be the anode.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Take one 50-mL beaker and a watch glass to the instructor&#39;s hood and obtain approximately 20 mL of acetone. Place the watch glass on top of the beaker to prevent evaporation and bring it back to your bench.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Label the other 50-mL beaker as &#39;organic waste&#39;.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a disposable pipette to rinse the copper electrode and key with acetone. Collect excess in the waste beaker. Place the key and copper electrode on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the initial masses of the key and copper electrode and record their values in your lab notebook.
		<h4><strong>Table 1: Moles of Cu Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td style="text-align: center;"><strong>Mass of key (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu plated </strong></td>
					<td style="text-align: center;"><strong>Mass of Cu electrode (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu lost </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">0 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_1._Moles_of_Cu_Transferred.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set up the electrolytic cell. Use one electrical wire with alligator clips to connect the negative terminal on the battery to the current probe. Wrap the 10-ohm resistor around the other end of the current probe.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use another wire to connect the positive terminal on the battery to the copper electrode.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Wrap the copper wire around the brass key and use an electrical wire to connect the resistor on the current probe to the copper wire on the brass key.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Obtain about 200 mL of concentrated copper sulfate in a 250-mL beaker. The amount does not need to be exact. Bring the beaker back to your bench and set it on the stir plate. Carefully, place a magnetic stir bar in the beaker and turn the stir setting on to high.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Connect the data acquisition system to the current probe and allow the data acquisition system to sense the probe. It should return a reading in amps. Change the rate to two scans per min and the duration to 5 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Place the brass key in the beaker. Hang it off the side of the beaker so that the key is almost completely submerged but does not touch the magnetic stir bar. <strong>Note: </strong>The copper wire should not be in the solution.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Place the copper electrode in the beaker, bending it so that it hangs over the edge of the beaker. Ensure that it is not touching the stir bar or the brass key.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Start the scan and let it collect data for 5 min. This will act as a test run to make sure plating occurs on the brass key.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">After the scan ends, remove the key and copper electrode. The copper plating on the brass key should be evident. If it is not visible, check your circuit to make sure that the battery is connected in the correct orientation.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once you have completed a successful 5-min plating, disconnect the key and copper electrode from the wiring. Remove the copper wire from the key and rinse the key and copper electrode with water and then acetone. Lay them on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Measure the masses of the key and the copper electrode and record them in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Reconnect the key and copper electrode and place them back in the solution like before. Restart the run on the data acquisition system, making sure that the rate is set to two scans per min. With the duration set to 30 min, allow the electroplating process to run.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">After the scan is complete, remove the electrode and key from the solution, disconnect them from the wires, and rinse them with water and then acetone. Allow them to dry on a paper towel, then weigh them and record their masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Reassemble the setup and repeat the electroplating for a second 30-min trial. When the scan is complete, remove the key and copper electrode, rinse them like before, and record their mass.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Save the recorded data as a text file on a flash drive.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">To clean up, turn the stir setting off and remove the stir bar from the solution using forceps. Dispose of the acetone in the organic waste container provided by your instructor. The copper sulfate solution can be reused for future labs, so pour the concentrated copper sulfate back into the carboy.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Rinse all of your glassware with water and return it to the instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, calculate the total charge Q that flowed through the system during the different trials. One coulomb is the quantity of electricity flowing through a wire carrying 1 ampere of current in 1 s, thus Q = I x t.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Review the current probe data for the first 5-min electroplating trial.<strong> Note: </strong>If the current dropped to 0 or close to 0 at the end of the trial, then the electrons stopped flowing from the copper electrode to the key. Only use the values recorded before the drop in current occurred. If the current drop to 0 early in the plating process, repeat the trial.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The current varies over time, so calculate the average current, I.
		<h4><strong>Table 2: Moles of Electrons Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>Time (s) </strong></td>
					<td style="text-align: center;"><strong>Current<sub>average</sub> (A) </strong></td>
					<td style="text-align: center;"><strong>Total charge (C) </strong></td>
					<td style="text-align: center;"><strong>Moles (e?) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">Total</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_2._Moles_of_Electrons_Transferred.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the average current over the total time to determine the total charge transferred during electroplating, Q. About 20 coulombs were transferred during the 5-min trial, and between 110 and 120 coulombs were transferred during each 30-min trial. The total charge transferred over the entire 65-min electroplating process was about 250 coulombs.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use the total charge transferred to calculate the moles of electrons plated during each trial. The moles of electrons plated equals the total charge transferred divided by the Faraday constant. The Faraday constant represents the magnitude of electric charge per mole of electrons and is determined by multiplying the charge of an electron by Avogadro&#39;s constant.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Determine the moles of electrons that were transferred for the 5-min and 30-min trials.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the moles of copper that were plated on the key after each trial. Similarly, determine the moles of copper lost from the copper sheet electrode.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the moles of copper that were lost from the electrode to the amount that was plated to the brass key. The numbers should be close.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check to see if these values match what is expected based on the number of moles of electrons that were transferred. The reduction of 1 mole of copper(2+) ions requires 2 moles of electrons to produce 1 mole of solid copper.</div>
		</li>
	</ul>
	</li>
</ol>

Electrolytic Cells: Construction and Electroplating - Procedure

電解セル

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Electrolytic CellsExpand
	In this experiment, you will be assembling an electrolytic cell to ...

JoVE Lab Manual

化学

Lab Manual Sub Articles

Synthesis of Luminol

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Synthesis of 3-Nitrophthalhydrazide<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll synthesize 3-aminophthalhydrazide, which is also called luminol, in a 2-step process. The first step is a condensation reaction between 3-nitrophthalic acid and hydrazine.</p>
	<p>During this step, the carboxylic acid groups are substituted with NH groups to produce 3-nitrophthalhydrazide and two water molecules. You must work in a fume hood throughout this lab because the reactions produce toxic vapors and gases.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. <strong>Note:</strong> Hydrazine is highly toxic and flammable, so avoid touching it and keep anything containing hydrazine closed or covered when you transport it between fume hoods.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Now measure 10 mL of deionized water and pour it into a 25-mL Erlenmeyer flask. Clamp the flask on a hot plate and place a thermometer in it.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Then, start heating the water. You&#39;ll want the temperature of the water to be 80 &#176;C, so set the hot plate setting to slightly higher.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">While it heats, set up a Bunsen burner under a clamp fixed on a lab stand.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Obtain a 25-mL test tube and adjust the clamp so that it will hold the test tube about 5 &#8211; 7 cm above the flame. Then, remove the test tube and place it in a 400-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Once the water reaches 80 &#176;C, adjust the heat setting to keep the temperature stable.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Then, weigh 0.6 g of 3-nitrophthalic acid in a tared weighing boat. Pour the 3-nitrophthalic acid into your test tube. Then, bring the test tube and a test tube stopper to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use the provided volumetric pipette to carefully measure 0.9 mL of the 10% v/v hydrazine solution and transfer it to the test tube. Remember to cap the bottle of hydrazine solution and stopper your test tube before you return to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Clamp the test tube over the Bunsen burner and remove the stopper. Then, pour ~2 mL of triethylene glycol into a graduated cylinder and set it out of the way in your hood.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Now, add 2 or 3 boiling chips to the test tube. Place a high-temperature thermometer in the test tube and ignite the Bunsen burner. Heat the mixture with a medium flame until the 3-nitrophthalic acid dissolves.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Then, add the triethylene glycol and increase the heat of the flame. <strong>Note:</strong> Once the water from the hydrazine solution has boiled off, the temperature will continue increasing.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">When the reaction mixture reaches 210 &#176;C, turn down the heat and keep the mixture between 210 &#176;C and 220 &#176;C for 3 &#8211; 4 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Then, extinguish the flame and wait for the mixture to cool to 100 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Turn off the hotplate, remove the thermometer, and unclamp the flask. Use tongs to hold the flask and measure 8 mL of hot water with the graduated cylinder. Pour this hot water into the test tube and remove the thermometer.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Obtain a piece of plastic paraffin film and cover the mouth of the test tube. Then, use tongs to transfer the test tube to the holding beaker.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">At a lab sink, hold the test tube under running tap water to cool it, being careful not to let water into it.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Then, dry the outside of the tube and bring it back to your hood. Clamp the tube upright and remove the paraffin film. Let the yellow 3-nitrophthalhydrazide precipitate in the tube for 15 min.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">While you wait, add ~10 mL of deionized water to a 25-mL Erlenmeyer flask. Prepare an ice bath in a 250-mL beaker and start chilling the water in it.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Then, set up for vacuum filtration using a 250-mL filter flask, a B&#252;chner funnel, and a piece of circular filter paper.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once the product has precipitated for at least 15 min, obtain a Pasteur pipette and wet the filter paper with a few drops of cold water.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Then, turn on the vacuum and pour the contents of the test tube into the B&#252;chner funnel. Rinse the remaining solid into the funnel with 1 &#8211; 2 mL of cold water.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Remove the boiling chips with tweezers and rinse them with a few drops of cold water to finish collecting the product.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Turn off the vacuum pump once no more liquid is dripping into the flask from the funnel.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Synthesis of 3-Aminopthalhydrazide<button class="expand">Expand</button>
	<p>In the second step of the luminol synthesis, you&#39;ll reduce the nitro group of 3-nitrophthalhydrazide using sodium dithionite in a basic solution of NaOH. This will produce 3-aminophthalhydrazide dianion.</p>
	<p>After the reduction, you&#39;ll add acetic acid to protonate the dianion, forming luminol. Sodium dithionite is highly reactive and degrades quickly, so you&#39;ll use a slight excess of it. After the reduction, you&#39;ll protonate the luminol dianion to decrease its solubility in water. Lastly, you&#39;ll precipitate and collect luminol as a yellow solid.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Place a clean 25-mL test tube in a beaker and use a clean spatula to transfer your 3-nitrophthalhydrazide to the test tube.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Set the B&#252;chner funnel aside and clamp the test tube over the Bunsen burner. Leave the filter flask in place for later.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, use a 10-mL graduated cylinder to obtain 3 mL of approximately 3 M NaOH. Pour the sodium hydroxide solution into the test tube and stir the mixture with a glass rod until the 3-nitrophthalhydrazide dissolves.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Now, weigh 2 g of sodium dithionite in a tared weighing boat and add it to the test tube. Obtain 3 mL of deionized water and pour it into the tube.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Stir the mixture well to dissolve as much of the solid as you can. Then, add a few clean boiling chips to the test tube and ignite the Bunsen burner.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Heat the mixture to boiling over a medium flame and then let it boil for 5 min.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">While you wait, measure 1.5 mL of glacial acetic acid and bring it to your hood.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Once the reaction mixture has boiled for 5 min, shut off the Bunsen burner and add the acetic acid to the tube. Let the mixture cool to room temperature, which usually takes 10 &#8211;15 minutes. Meanwhile, prepare an ice bath in a 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the mixture has cooled, place it in the ice bath and wait 15 min for the luminol to precipitate.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Reassemble the vacuum filtration setup using a clean B&#252;chner funnel and a new piece of filter paper, and start cooling another 10 mL of deionized water in an ice bath.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once the luminol has been in the ice bath for at least 15 min, wet the filter paper with cold water. Then, collect the solid luminol by vacuum filtration.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Retrieve the boiling chips and rinse the luminol with more cold water. Once liquid stops dripping from the funnel, turn off the vacuum and transfer the luminol to a 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Now, prepare the combined filtrate for disposal. First, label a 250-mL beaker &#8216;hydrazine and dithionite waste&#8217;. Transfer the filtrate to the beaker and rinse the flask with deionized water to remove residual hydrazine and dithionite.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Next, dilute the filtrate with 10 mL of deionized water. Then, measure 10 mL of 1 M sodium carbonate and add it to the combined filtrates.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Bring a 50-mL graduated cylinder to the waste hood. Measure 40 mL of 10% sodium hypochlorite and pour it into the filtrate mixture. Place the beaker on the hot plate, add a stir bar, and secure a thermometer in the beaker.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Heat the mixture to 50 &#176;C while stirring. The mixture must stay at 50 &#176;C for 1 h to oxidize leftover hydrazine and dithionite, so make sure that the temperature is stable and set a timer for 1 h before starting the last part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Chemiluminescence and&#160;Fluorescence<button class="expand">Expand</button>
	<p>For the last part of the lab, you&#39;ll mix some of the luminol you made with dimethyl sulfoxide over solid potassium hydroxide. The hydroxyl ions deprotonate the two amine groups. Then, ambient oxygen oxidizes the luminol dianion to 3-aminophthalate, or 3-APA, in an excited state. This unstable complex will quickly relax to the ground state, releasing energy as visible light. Since the excited 3-APA was the product of a chemical reaction, the emitted light is called chemiluminescence.</p>
	<p>Once you have seen the chemiluminescence from the oxidation reaction, you&#39;ll add fluorescein, a fluorescent molecule, to the mixture. Some excited 3-APA will transfer energy directly to fluorescein rather than emitting light, giving you a solution with two different light-emitting compounds.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-3">
		<div class="lab-step">Now, measure about 0.2 g of the luminol you made and place it in a 15-mL Erlenmeyer flask. It&#39;s OK if the solid is still damp.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Obtain 5 &#8211; 7 pellets of potassium hydroxide and pour them into the flask. Then, bring the flask, a stopper, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Measure 2 mL of DMSO, add it to the flask, and stopper it. Put the graduated cylinder in your fume hood and bring the flask to a dark room.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Shake the stoppered flask vigorously until you see chemiluminescence and then put the flask down. The light fades quickly because both the oxidation and relaxation are fast processes.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Shake the flask and let the light fade 6 &#8211; 10 more times without opening the flask. The light will be progressively dimmer as the oxygen in the flask is consumed. <strong>Note:</strong> To make it brighter, leave the flask open in your fume hood for a minute to replenish the oxygen.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Close the flask, return to the dark room, and shake the flask again to see the effect.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Now, measure 0.2 g of fluorescein dye and add it to your flask. Stopper the flask and return to the dark room. Shake the flask vigorously until the mixture is glowing brightly and then set it down to observe the difference in color from luminol alone.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">When you&#39;re finished, check on the filtrate. Once it has been at 50 &#176;C for at least 1 h, turn off the hotplate and wait for the beaker to cool to room temperature.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Then, add about 10 mL of tap water to the filtrate and flush it down the drain.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Dispose of the remaining chemical and glass waste in the appropriate containers, clean your glassware and tools, and put away your lab equipment.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Under your oxidation reaction conditions, excited 3-APA emits blue-green light in a broad range around 500 nm when it relaxes, and fluorescein is excited by absorbing light at 480 &#8211; 490 nm and 515 &#8211; 525 nm.</p>
	<p>When you add fluorescein to the luminol oxidation reaction mixture, excited 3-APA can transfer energy that fluorescein would absorb as light directly to a nearby fluorescein molecule in a special interaction called nonradiative energy transfer. The resulting excited fluorescein emits yellow-green light when it relaxes.</p>
	<p>Excited 3-APA is highly unstable, so if no fluorescein is close enough for nonradiative energy transfer, it will relax by emitting light as usual. Thus, a spectrum of your final glowing mixture would show contributions from both blue-green and yellow-green light.</p>
	<ul class="lab-items">
	</ul>
	</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Procedure

ルミノールの合成

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Synthesis of 3-NitrophthalhydrazideExpand
	In this lab, ...

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Preparing 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7</strong><button class="expand">Expand</button>
	<p>In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.</p>
	<p>Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before starting the experiment, calculate the mass of monosodium phosphate that you will need to make 200 mL of a 50 mM solution.
		<h4><strong>Table 1: Preparation of 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7.0</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="247" width="358">
			<tbody>
				<tr>
					<td colspan="2"><strong>Molar mass of NaH<sub>2</sub>PO<sub>4</sub> = 119.98 g/mol</strong></td>
				</tr>
				<tr>
					<td>Solution volume (mL)</td>
					<td style="text-align: center;">200</td>
				</tr>
				<tr>
					<td>Molarity (mM)</td>
					<td style="text-align: center;">50</td>
				</tr>
				<tr>
					<td>Moles of NaH<sub>2</sub>PO<sub>4</sub> (mol)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass needed (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Initial volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Final volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of NaOH used (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of DI water needed (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_1._Preparation_of_50_mM_NaH2PO4_Buffer,_pH_7.0.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, put on the necessary personal protective equipment, including a lab coat, splash-resistant safety glasses, and gloves. <strong>Note: </strong>This section of the lab uses NaOH, which is corrosive and toxic. Use caution when pouring and transporting NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Fill a 250-mL plastic wash bottle with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label two 100-mL beakers, one for neutral aqueous waste and one for basic aqueous waste.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Tare weighing paper, and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Record the exact amount of monosodium phosphate in your lab notebook. <strong>Note: </strong>Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement and close the container tightly when you are done with it.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place the monosodium phosphate in a 400-mL beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a graduated cylinder to measure out 175 mL of deionized water. Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Stir the solution until the salt has dissolved completely and the solution appears homogeneous. This usually takes 2 - 3 min.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 15 mL of deionized water and pour it into the beaker. Continue stirring the solution until it appears homogeneous again (about 1 &#8211; 2 min). Turn off the stir motor.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Bring a 10-mL graduated cylinder and watch glass to the dispensing hood and measure out 10 mL of 1 M NaOH. Note the exact volume in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Cover your NaOH with the watch glass, and carefully bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add NaOH to the stirring solution in a drop-wise manner. Once the buffer pH reaches 7.0, return any NaOH still in the pipette to the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Calculate the volume of NaOH that you added to the buffer. Subtract that volume from 10 mL to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 mL.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure the deionized water with another 10-mL graduated cylinder and add it to the stirring solution to finish making the buffer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Label a 250-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer, pH 7.0&#39;. Retrieve the magnetic stir bar from the solution.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Use a funnel to pour the buffer solution into the labeled bottle and cap it tightly.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Absorption Spectroscopy of Free and Bound Neutral Red</strong><button class="expand">Expand</button>
	<p>Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.</p>
	<p>After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Draw the following table in your lab notebook. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use. The 10th cuvette will be a deionized water blank. <strong> Note: </strong>Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
		<h4><strong>Table 2: Absorbance of Free and Bound Neutral Red</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Cuvette #</strong></td>
					<td style="text-align: center;"><strong>Buffer pH </strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max </sub>(Free NRH<sup>+</sup>)</strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max</sub> (Bound NRH)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">5.0</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">5.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">6.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">6.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">7.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;">7.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;">8.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;">8.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;">11</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;">blank</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_2._Absorbance_of_Free_and_Bound_Neutral_Red.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Obtain ten 1.5-mL cuvettes and caps and label the caps 1 through 10 to match the table in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 25-mL beaker as &#8216;DIH2O&#8217; and fill it with deionized water.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Flush your neutral aqueous waste down the drain and relabel the beaker as &#39;aqueous buffer waste&#39;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker for used micropipette tips.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Attach a tip to a 1-mL micropipette and use it to dispense 1000 &#181;L of deionized water into cuvette 10. This is your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 925 &#181;L of your pH 7.0 monosodium phosphate buffer in cuvette 5.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 &#181;L of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once you have finished, bring the buffers back to your workbench. There, set a 200-&#181;L micropipette to dispense 75 &#181;L, and attach a tip to the micropipette.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Dispense 75 &#181;L of neutral red into each of the nine buffer cuvettes. Invert each cuvette several times to thoroughly mix the solutions. <strong>Note: </strong>Replace the pipette tip if it touches a buffer.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Turn on a hand-held spectrophotometer and allow the light source to warm up. Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Insert the cuvette of deionized water and acquire a spectrum of the deionized water. Set it as a solvent background or blank.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Then, remove the blank and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum. Record this wavelength in your lab notebook as lambda max for protonated free neutral red.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Save the data and remove the cuvette. Record the absorbance in your notebook, then collect spectra for cuvettes 2 - 9 using the same procedure.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Record the wavelength of the isosbestic point in your lab notebook. That is where all the spectra cross at a single wavelength. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Once you finish collecting spectra for all nine cuvettes, save and export the data.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Create a new experiment to measure absorbance versus wavelength for the bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Fit a new tip to the 200-&#181;L micropipette and add 75 &#181;L of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Insert cuvette 10 into the spectrophotometer and set it as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Acquire a spectrum of the lowest pH sample, and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Acquire spectra for cuvettes 2 - 9 in the same way as you did for the free neutral red samples. Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">When you are done, save your data, export it for later analysis, and shut down the spectrophotometer.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Put away all equipment and dispose of your used pipette tips in an approved waste container or trash bin.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Empty the excess NaOH into the basic waste and rinse the graduated cylinder with water.</div>
		</li>
		<li data-sub-note-item="2-30">
		<div class="lab-step">Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste.</div>
		</li>
		<li data-sub-note-item="2-31">
		<div class="lab-step">Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<p>Now, let&#39;s analyze our absorbance data to determine the pKa&#39;s of neutral red.</p>
	<h4><strong>Table 3: Determination of Neutral Red pKa&#39;s</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td></td>
				<td>Free NRH<sup>+</sup></td>
				<td>Bound NRH<sup>+</sup></td>
			</tr>
			<tr>
				<td>&#160;&#955;<sub>max</sub> (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;&#916;A (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;Midpoint (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;pKa</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/45/Table_3._Determination_of_Neutral_Red_pKa.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Plot both sets of intensity data with absorbance intensity at lambda max on the y-axis and pH on the x-axis, with the points joined by smooth lines.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the difference between the starting and ending absorbance intensities for each data series.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Determine the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Find where the midpoint occurs on each line and identify the pH value at that point. These pH values are the pKa&#39;s of free and bound neutral red.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Here, we see an increase in pKa of about one between free and bound neutral red, indicating that bound-protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Procedure

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Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparing 50 mM NaH2PO4 Buffer, pH 7Expand
	In the first part of this experiment, you will ...

Scientific Measurement and Lab Skills

Two-Dimensional Graphs, Curve Fitting, and Standard Deviation - Procedure

科学的な測定とラボスキル

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Measuring Diameter and CircumferenceExpand
	Error can be introduced in any experiment, even ...

Optimal Foraging

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Foraging with Artificial Habitats and Prey
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with varying prey densities. Without knowing what these densities are, foragers must obtain as many prey items as possible in as little time as possible. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">HYPOTHESES: In this experiment, the experimental hypothesis could be that foragers will catch the most prey in the higher prey density bucket and also spend the most time foraging there.  The null hypothesis would then be that foragers will catch an equal number of prey in each bucket and that foragers will spend an equal amount of time foraging in each patch. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To begin the foraging activity, record the names and role of each participant at the top of the student data collection sheet.  NOTE: Before starting the experiment, the forager is allowed to become familiar with the foraging process &#8211; the following steps are a practice round. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">The forager should kneel or sit in front of the average density bucket. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Once the timer says start, the forager may begin looking for the pinto beans representing the prey. The forager can spend as much time as they like foraging in this bucket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once a prey item is located, the forager must signal that one has been found and place the prey in the cup next to the bucket.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once they believe they have acquired most of the prey in the patch, they should tell the timer to stop the time and the practice round is over. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the simulation proper, the forager should sit or kneel in front of bucket A. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The timer must be ready to start the stopwatch. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The recorder should be ready to record the time each prey item is located, in addition to the time that the forager arrived at and left each bucket. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When all parties are ready, the timer should start the stopwatch and say &#8220;start&#8221;,  and the forager should begin foraging. NOTE: The stopwatch will run through the rest of the round for buckets B and C.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">As the forager signals finding pinto beans and places them in the cup next to the bucket, the timer should tell the recorder the time.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">The recorder should then write the time in the student data collection sheet.  <a href="https://www.jove.com/CDNSource/lm/labs/34/Table_1_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Once the forager has decided to move on to the next bucket, they should tell the timer &#8220;done&#8221;, and begin moving to the next patch. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">The recorder should record the time the forager left and the time they arrived at bucket B in the student data collection sheet.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Repeat the same procedure for buckets B and C. Once the forager has completed the activity, it is time for the next forager to begin using the same procedure. 
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Before performing any data analysis, review the data collection sheet and compile your individual data for captured prey, foraging time, and travel time for each bucket in your student calculations table. <a href="https://www.jove.com/CDNSource/lm/labs/34/Table_2_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">After this, calculate your individual capture rate for each bucket by dividing the number of prey captured by the sum of the foraging time and travel time and record these values in your table. Individual capture rate =  Number of prey captured / (foraging time + travel time)
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To calculate your Giving-Up-Time or a GUT for each bucket, subtract the time that the last prey item was acquired from the time you left the bucket.  GUT = time forager left bucket  -  time last prey item acquired
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, calculate the mean GUT for your foraging group and record the values in your table.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Finally, calculate your scaled GUT for each bucket by dividing the bucket GUT by your group&#39;s mean GUT. Scaled GUT =  Bucket GUT / Group mean GUT NOTE: The scaled GUT will allow class data to be pooled later on without differences in individual foragers skewing the data. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">The instructor should now compile the data into the classroom spreadsheet.  <a href="https://www.jove.com/CDNSource/lm/labs/34/Table_3_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Plot a bar graph of the average number of prey collected in each bucket. On the x-axis, A should represent the low prey density bucket, B should represent the medium prey density bucket, and C the high prey density bucket. Plot the y-axis as the number of prey items caught. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, plot the graph of the class average scaled GUT, where the x-axis represents the buckets with varying levels of prey density and the y-axis shows the values for the average scaled GUT. 
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Plot a third graph of the average time spent foraging in each bucket. 
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Now review the Marginal Value Theorem (MVT) from the concepts video and examine the three graphs that you generated from your class data. Take note of whether the graphs conform to the predictions of MVT. 
</div></li></ul></li></ol>

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

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Simulating Foraging with Artificial Habitats and Prey
ExpandNOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with ...

生物学

生態学

Cellular Respiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quantifying Respiration using Microrespirometers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment, you will measure the rate of cellular respiration for germinating seeds by measuring the rate of exchange for oxygen. As oxygen is consumed to provide energy, germinating seeds release carbon dioxide. This carbon dioxide is absorbed by potassium carbonate and thus the overall gaseous pressure of the respirometer will be reduced. HYPOTHESES: The experimental hypothesis is that germinating seeds will show a greater rate of respiration than control glass beads. Additionally, that at higher temperatures, the rate of cellular respiration in the seeds will increase. The null hypotheses are that both glass beads and germinating seeds will display a similar rate of cellular respiration and that temperature has no effect on respiration.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To set up the control, first remove the plunger from a prepared microrespirometer and label it with a &#8220;CR&#8221; or &#8220;CH&#8221; respectively, depending on whether it is the control room temperature or heated treatment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Add the glass microbeads to each of the microrespirometers up to the 0.5 mL mark, and push the plunger in the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.  NOTE: The microrespirometer will sit capillary tube end up.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the control devices to the side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To set up the experimental seated respirometers, remove the plunger from a prepared microrespirometer and label it with &#8220;ER&#8221; or &#8220;EH&#8221; respectively, depending on whether it is the experimental room temperature or heated treatment.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Measure 0.5 mL of germinating seeds into the tuberculin syringe of the microrespirometer.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, push the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">To use the microrespirometers, carefully press the &#8220;CR&#8221; and &#8220;ER&#8221; microrespirometers into the room temperature water bath. The entire syringe should be submerged, but the capillary tubes should be entirely exposed. Remove water from the bath until this position is achieved. Be sure the top of the capillary tube is open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Perform the same procedure (step 9) for the &#8220;CH&#8221; and &#8220;EH&#8221; microrespirometers to set up the higher temperature experimental group.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Wait 5 minutes before moving on to allow the temperature in the microrespirometers to equalize with the water bath.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Add a single drop of manometer fluid to the top of each of the four capillary tubes which seals the microrespirometer chambers.  NOTE: If the chambers are working properly, the manometer fluid will be sucked into the capillary chamber.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Use the plunger set in the bath to suck the manometer fluid into the capillary. Stop when the fluid is about halfway down the capillary tube. IMPORTANT: The microrespirometers should not be disturbed once they are added to the bath. Do not bump the table or insert or remove anything to or from the water bath once the chambers have been sealed and equalized.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Mark the bottom edge of the manometer fluid that is closer to the chamber. This represents time point zero.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Set a timer to sound in 5 minutes.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After 5 minutes, create a new mark where the manometer fluid is on each microrespirometer. Repeat every 5 minutes until 25 minutes has passed or until the manometer fluid has traveled the entire length of the capillary tube.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, remove the respirometers from the water baths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Measure the distance between each of the marks and record them in Table 1 next to the correct time point. <a href="https://www.jove.com/CDNSource/lm/labs/8/Table_1_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>  
</div></li><li data-sub-note-item="1-20"><div class="lab-step">During clean up, manometers should either be deconstructed to be reused or disposed of in the trash.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To analyze the data you collected, first plot the data points with the x-axis as time and the y-axis as the distance the manometer moved, representing oxygen consumption.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Calculate the slope of each line using this equation.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record these values for each microrespirometer in Table 2. NOTE: This value represents the amount of cellular respiration that took place over the course of the experiment.  <a href="https://www.jove.com/CDNSource/lm/labs/8/Table_2_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Plot the respiration rates as a bar graph.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Examine both of the graphs. Consider how your results fit with the hypotheses. Note any observed differences in respiration rate between the control beads and the germinating seeds. Determine if increasing the temperature had any effect on the respiration rate.
</div></li></ul></li></ol>

Cellular Respiration: Quantification using Microrespirometers - Procedure

細胞呼吸

Quantifying Respiration using Microrespirometers
ExpandNOTE: In this experiment, you will measure the rate of cellular respiration for germinating seeds ...

細胞プロセス

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

動物の行動

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

Extinction

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Diversity and Extinction during the Paleogene Epoch
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin this procedure by reading the extinction reading supplement that has been printed out for you. <br /><strong>NOTE:</strong> Alternatively, you may download the reading supplement at the link below.&nbsp; <a href="https://www.jove.com/CDNSource/lm/labs/24/Lab_20_Reading Supplement.docx" style="display:block;margin:20px;" target="_blank">Click here to download the extinction reading supplement</a>
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Using the information contained in the extinction reading supplements pertaining to the taxa of interest and the Chicxulub impact event, make predictions for each taxon listed in Table 1. Consider whether diversity in each taxon increased, decreased, or stayed the same immediately following the Cretaceous-Paleogene boundary and later on in the Paleogene epoch. <a href="https://www.jove.com/CDNSource/lm/labs/24/Table_1_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, fill out the reasons for each of your predictions in the Contributing Factors column. HYPOTHESES: The experimental hypothesis is that many of the different taxa will show changes in diversity after the Cretaceous-Paleogene event, and again at the end of the Paleogene, with some increasing in diversity, while others decrease. The null hypothesis is that there will be no changes in diversity between taxa following the Cretaceous-Paleogene event, or at the end of the Paleogene.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Gather diversity data by going to the Paleobiology Database: <a href="http://www.paleobiodb.org" target="_blank">www.paleobiodb.org</a>.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In the center of the screen, click the yellow box labeled Download Data. Under the italicized heading, &#8220;What do you want to download?&#8221;, select Diversity over time on the left side and Comma-separated values on the right side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Scroll down to the heading &#8220;Select by taxonomy&#8221;.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the first box labeled 'Taxon or taxa to include', type in the name of the taxon that data is being gathered on.  NOTE: Ensure the spelling is correct.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Now, go to the dropdown menu right below that box labeled &#8220;Taxonomic resolution&#8221;, and select genera.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Under the next heading, Select by time, type Maastrichtian into the first box and Chattian in the box directly below. Make sure the dropdown menu labeled Time rule is set to major and the Temporal resolution is set to stage.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Select &#8220;Assume extant taxa range through to present&#8221;. Scroll back up to the middle of the page and click download. A save window will open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Save the .csv file to a safe and accessible place and rename it to make it easy to identify.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Open the file and scroll down to the main dataset under Records.  NOTE: The data are arranged in reverse time order by geological stage. The second column, interval_name, gives the accepted name of the stage in question. The next two columns give the appropriate dates of the stage in millions of years before the present. The final column, number of occurrences, gives a total count of the genera extant during that geological stage as determined from occurrence of fossils.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To more easily work with this data, copy and paste each column into a new sheet. Be sure to label this sheet with the taxon in question, the data and time the data were obtained, and the data source.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat this process for each of the taxa in Table 1 (steps 4 &#8211; 12). NOTE: Data from multiple taxa can be kept on the same sheet, so long as they are kept distinct and labeled appropriately.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To create line plots for each of the taxa showing the change in diversity over time, select the max_ma column and the number of occurrences column simultaneously.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">In Excel, select Insert from the top toolbar and find the section labeled Charts. Select the bottom center icon, Insert Scatter Chart.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, select the Scatter with Straight Lines and Markers and a graph should appear. NOTE: Since the axis of this graph is in units of millions of years ago, the axis is in the reverse direction of time when put in numerical order.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">To fix this, click on the green plus sign in the top-right corner of the graph and hover over Axes. This should reveal a small black arrow. Click the arrow to open up a small menu and select More Options.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">This will open the Axis Options menu. Under Axis Options, click the box next to Values in reverse order. NOTE: It is also possible to change the axis scales from this menu, which can be used to eliminate the excess time that is not being analyzed in this lab. Keep any changes consistent between different graphs.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Double-click on the title at the top of the graph and change it to the taxon being represented.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Make a separate graph for each of the taxa in Table 1. Keep them organized and in one location. A separate sheet for graphs is recommended.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">For each graph, identify the location of the Cretaceous-Paleogene boundary and draw a vertical line through the graph at this point.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using the information from the graphs, fill out Table 2. In some cases, it may be helpful to go back to the original dataset and compare actual values, particularly when they are small. <a href="https://www.jove.com/CDNSource/lm/labs/24/Table_2_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare these values to the predictions made in Table 1 and discuss if the original contributing factors are still likely candidates.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Using the dataset, calculate the percentage of total taxa that went extinct over the Cretaceous-Paleogene boundary using this equation.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">There is an event in the middle of the Paleogene known as the Paleocene-Eocene Thermal Maximum about 55.5 million years ago. This represents a high point in temperature during the Paleogene epoch at approximately 8 &#176;C higher than today&#39;s average temperature. Does this point seem to have an effect on diversity for any of the taxa? If yes, then which ones?
</div></li></ul></li></ol>

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

絶滅

Diversity and Extinction during the Paleogene Epoch
ExpandBegin this procedure by reading the extinction reading supplement that has been printed out for ...

Measuring Biodiversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quadrats and Transects
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this activity, you will be using quadrats along transect lines to take biodiversity measurements of the plant species in three distinct habitats near your classroom. Begin by splitting into groups. Four works best.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Select one person in your group to be the data recorder. The other three will be the data collectors.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, take one quadrat, or hula hoop, per group and place it on one side of one transect, or rope, next to a knot to allow identification of the plant species encircled by your quadrat. HYPOTHESES: For this experiment, the alternative hypothesis is that the alpha diversity will be smaller than gamma, meaning that there will be differences in the species that are present in each different habitat. The null hypothesis for this activity is that the alpha and gamma will be equal, meaning that there will be no species diversity difference between the communities sampled.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Each data collector must survey and identify the different plant species or any morphologically distinct varieties within your group&#39;s quadrat, giving the name of each plant type that you identify to the data recorder. It may seem like there are only a couple of species present at first, but look closer. It may take several minutes to find them all. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In addition to recording the species name, the data recorder must also write a short description in the table, so that each species can be identified again in subsequent quadrats. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_1_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once all of the species within a quadrat have been identified, pick up your hoop and move on to the next knot along the same transect.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Continue surveying and moving to the next knot until you have completed the entire rope transect.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Once you have completed the rope, move to a transect in a different habitat, and begin identifying the different species within each quadrat along the new transect, until each group has identified plants along three lengths of rope, one in each habitat, for a total of 15 quadrats surveyed.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">When all of the species data have been collected, carry the transects and quadrats back to the classroom.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Species-Accumulation Curves and Richness/Evenness 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">In the next activity, you will be calculating and comparing the species richness and evenness in two distinct populations, represented here by two opaque bags containing beads of various colors. For this exercise, start by taking one paper bag of beads per group.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Have one person reach into the bag and randomly pull out three beads without looking.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Write down the bead colors, as well as the total number of colors pulled from the bag in the table. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_2_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>HYPOTHESES: In this activity, the alternative hypothesis states that species evenness in the two communities will be different, while the richness will be the same. The null hypothesis might be that species richness and evenness will be the same in both communities.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Place the beads back in the bag after their colors are recorded.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, shake the bag and have the next person in the group draw another set of three beads.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record the colors and numbers of beads that they pulled in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Record the colors in the second row and the cumulative number of colors drawn in the last column.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Continue to take turns drawing three beads from the bag until 20 samples have been drawn.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Next, switch bags with a group with a bag from the other community, and repeat the exercise of drawing 20 samples, recording the color data in the appropriate community column of the table.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">When all of the data have been recorded, return the beads to the bag and return the bags to your instructor.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To calculate the alpha diversity of the transect and quadrat data collected by your group, use the data your data recorder entered for each species and enter it into your diversity table. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_3_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, fill out the alpha diversity for each habitat into the appropriate column in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Use the species data from your quadrat and transect table and enter the beta diversity data into the appropriate data cells within the diversity table. NOTE: The beta diversity is the number of species that are unique between two habitats. In this video, there are three species in common between the two sites, but a total of six unique species, giving a beta diversity of six between site A and B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">To calculate the gamma diversity for your activity, use the data from your quadrat and transect table and enter the gamma diversity data into the appropriate data cell. NOTE: The gamma diversity is the number of species in all of the habitats within a single study. It is not the sum of all of the alpha diversities, because species that are common between habitats should be counted only once.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">To construct species accumulation curves for both of the bead sampling communities, use your bead color selection data to plot the species by sampling effort. That is the number of total colors by sample in the graph for each bead bag community.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, estimate the asymptote of the plotted data by appearance. Predict the number of species you would expect to find in each community. Note any differences in appearance between the two curves. If it was easier to estimate the number of species in one of the communities, make a hypothesis to explain this observation.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">To construct rank abundance curves for both of the bead sampling communities, use the color bead data to fill in the table for both community A and community B, listing the colors that were identified during the sampling in the color column, and adding the total number of beads of each color that was sampled in the abundance column. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_4_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 4</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Now, assign a rank to each color in the rank column, designating the color of the highest abundance the highest rank, rank one, and the color with the lowest abundance the lowest rank, rank 10. If there are multiple colors with the same number, just assign them consecutive ranks.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Plot individual rank abundance curves for the number of individuals by species ranked for the most to least common in both community A and community B. Compare the steepness of the two rank curves. Compare the richness and evenness between the two communities. Identify the community that you consider more biodiverse. 
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生物多様性の測定

Quadrats and Transects
ExpandIn this activity, you will be using quadrats along transect lines to take biodiversity measurements of the plant species in ...

Scientific Method

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observation and Background Research
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The first two steps in the scientific process involve studying and observing natural phenomena of interest.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Observe the leaves and branches of the individual plant species. Each species comes from a different environment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Compare each branch to one another and record your observations about any qualitative similarities or differences. <a target="__blank" style="display:block;padding:20px" href="https://www.jove.com/CDNSource/lm/labs/1/Table_A_Lab_1.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use leaf clippers to cut the leaves from the branches.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Make additional qualitative observations about the leaves from each species.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Hypothesis Formulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Think of some general questions that you might have after observing the different plant species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these general questions, create a null and an experimental hypothesis.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Experimentation and Data Collection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Weigh and record the mass of each leaf. <a target="__blank" style="padding:20px;display:block;" href="https://www.jove.com/CDNSource/lm/labs/1/Table_B_Lab_1.xlsx">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Measure and record the length of each leaf from the petiole to the tip in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next, carefully trace each leaf outline onto a blank piece of computer paper.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, cut out and weigh and 4 x 4 cm square of the same paper, weigh the cutout tracing, and record these data in the table.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Data Analysis and Conclusion
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">To calculate the leaf surface area, divide the surface area of the 4 x 4 cm paper square by the mass of the paper square, and multiply the resulting number by the mass of the leaf tracing.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis, and the surface area in square centimeters indicated on the y-axis.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Draw a trend line through the graph if a relationship is evident.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Using your qualitative observations and quantitative measurements, draw conclusions about the data you collected.
</div></li></ul></li></ol>