Name: exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal
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<p class="jove_content" style=";text-align:right;direction:rtl"> المؤلفون تعترف مع الامتنان المساعدة الماهرة السيدة ترين W. ميكلسن، السيدة ليز M. المناسب، والموظفين في Påskehøjgaard. المجلس الدانمركي للأبحاث الطبية، ومؤسسة وندبيك، ومؤسسة نوفو نورديسك الدعم المالي للدراسة. </p>

<p class="jove_content" style=";text-align:right;direction:rtl"> تم إجراء تخدير الحيوان وeuthanesia وفقا ل &quot;مبادئ مختبر رعاية الحيوان&quot; (NIH المنشور رقم 86-23، منقحة 1985) والتي وافق عليها المجلس الدانمركي لأخلاقيات البحوث الحيوانية. </p><p class="jove_title" style=";text-align:right;direction:rtl"> 1. أدوات </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع الأدوات المعروضة في الفيديو والمدرجة في <strong>جدول المواد.</strong> </li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 2. قطع الرأس </p><p class="jove_content" style=";text-align:right;direction:rtl"> وكان المستحث التخدير عن طريق الحقن العضلي من 5 مل من ميدازولام (5 ملغ / مل) و 5 مل من الكيتامين (25 ملغ / مل): ملاحظة. في وقت لاحق 5-10 دقيقة، عندما كان مخدرا للحيوان بالغ، ومقنى الوريد الأذن وجرعة زائدة قاتلة (100 ملغم / كغم من وزن الجسم) من الصوديوم بنتوباربيتال أعطيت (200 ملغ / مل) عن طريق الوريد. للتأكد من أن هذا الحيوان كان الموت الرحيم تماما، تم اختبار رد الفعل الألم بين الأصابع كما يتضح من Ettrup <em>وآخرون.</em> (2011) <sup class="xref">20.</sup> وقد ضمنت euthanization الكامل كما هو موضح فيبيان الأخلاق أعلاه ويليه نضح transcardial مع 5 L من المياه المالحة متساوي التوتر، كما يتبين من Ettrup <em>وآخرون.</em> (2011) <sup class="xref">20.</sup> يتم تنفيذ كافة الإجراءات أثبت تشريح الجثة، والحيلولة دون الحاجة إلى الاحتياطات الرعاية اللازمة للتخدير طويلة الأجل والبقاء على قيد الحياة postprocedural. </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع رأس خنزير بواسطة شق عنق الرحم دائرية عالية، وذلك باستخدام مشرط جراحي، أقل بقليل من زاوية الفك السفلي <strong>(الشكل 1A).</strong> </li><li style=";text-align:right;direction:rtl"> لا يزال مع مشرط جراحي، ومواصلة شق الأمامية من خلال الأنسجة الرخوة في الرقبة، بما في ذلك الحنجرة والمريء، حتى يتم التوصل إلى العمود الفقري عظمي، تقريبا على مستوى تقاطع craniocervical. </li><li style=";text-align:right;direction:rtl"> تقدم قطع مع مشرط جراحي من الجانب الأمامي من مفترق الطرق craniocervical، فوق قوس الأمامي من الأطلس، وذلك من خلال الغشاء الفهقي الأمامي، وبالتالي تعريض القناة الشوكية والحبل الشوكي (<strong>الشكل 1B).</strong> نسأل نفس الوقت مساعدا لسحب الجسم الخنازير بعيدا عن رأس خنزير لتخفيف الوصول بين قاعدة الجمجمة وأول فقرة الرقبية. </li><li style=";text-align:right;direction:rtl"> مواصلة شق جراحي من خلال الكيس الجافية والحبل الشوكي <strong>(الشكل 1B).</strong> عناية خاصة لضمان أن يتم التوصل إلى المقطع العرضي كاملة من الحبل الشوكي. <br /> ملاحظة: الفشل في تنفيذ الخطوة السابقة قد يؤدي إلى جر غير المرغوب فيها على الحبل الشوكي والدماغ خلال الخطوات التالية لعملية قطع الرأس. </li><li style=";text-align:right;direction:rtl"> تمديد بقوة تقاطع cranocervical على مستوى القسم <strong>(الشكل 1C).</strong> في نفس الوقت، استخدم مشرط جراحي لقسم الأربطة الفهقي المتبقية للافراج عن التعبير بين لقمة القذالي وعملية التعبير العليا من الأطلس. فصل رأس خنزير من الجسم. </li></ol><p style=";text-align:right;direction:rtl"><img alt="شكل 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">الشكل 1: Minipig قطع الرأس.</strong> <strong>(A)</strong> العنق شق (السهم، زاوية الفك السفلي). <strong>(B)</strong> شق طريق الأربطة الفهقي والنخاع الشوكي محاط الجافية (SC) عند تقاطع craniocervical (C1، القوس الأمامي من أطلس، OC، لقمة القذالي). يتم تحريرها <strong>(C)</strong> والجزء الخلفي من التعبير الفهقي بواسطة ملحق قوية (الأسهم) على مستوى القسم. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> </p><p class="jove_title" style=";text-align:right;direction:rtl"> 3. الجمجمة افتتاح </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع رأس خنزير على الطاولة. </li><li style=";text-align:right;direction:rtl"> إجراء شق طولي الظهري مع مشرط جراحي عن طريق الجلد والأنسجة الرخوة الكامنة من الجزء الخلفي من الخطم، على قمة الرأس، وأسفل من خلال الجزء الخلفي منالمنطقة القفوية. </li><li style=";text-align:right;direction:rtl"> كشف ظهري والخلفية جزء من الجمجمة عن طريق إزالة الأنسجة اللينة تقع الوحشي على شق الأولي مع مشرط جراحي. </li><li style=";text-align:right;direction:rtl"> الافراج عن العضلات الزمنية ثنائيا من الجمجمة <strong>(الشكل 2A)</strong> مع مشرط جراحي. تأكد من أن عظم القذالي الخلفي يتم تنظيف الأنسجة اللينة. </li><li style=";text-align:right;direction:rtl"> استخدام المدخل الخلفي للماغنوم الثقبة لإزالة عظم القذالي مع Kerrison العظام لكمة والعظام رينجرز وفضح المخيخ المغطاة الجافية <strong>(الشكل 2B).</strong> </li><li style=";text-align:right;direction:rtl"> العودة إلى الجانب الأمامي المكشوفة من الجمجمة وتحديد نقطة الدخول في العظم الجبهي، عادل امام العينين. في هذه المرحلة، واستخدام إزميل العظام بمطرقة لاختراق الجمجمة وأدخل الجيب الأمامي <strong>(الشكل 2C).</strong> </li><li style=";text-align:right;direction:rtl"> استخدام مدى الجيب الأمامي لمواصلة إزالة ظهراني خلفي من الصفيحة الجمجمة الخارجي مع مقراض العظم أو العظام لكمة وفضح الداخلية، رقيقةالعظمية الصفيحة الجمجمة التي تغطي المخ <strong>(الشكل 2D).</strong> </li><li style=";text-align:right;direction:rtl"> فتح بلطف العظمية الداخلية الصفيحة الجمجمة الأمامية مع مطرقة وإزميل العظام لفضح المخ المغطاة الجافية <strong>(الشكل 2E).</strong> </li><li style=";text-align:right;direction:rtl"> تواصل إزالة العظام أفقيا باستخدام إزميل العظام ومقراض العظم من خلال العظم الصدغي والجداري من أجل الإفراج عن الجزء ظهراني خلفي النهائي من الجمجمة، وتقع بين الأجزاء المكشوفة بالفعل للمخ المغطاة الجافية والمخيخ <strong>(الشكل 2F).</strong> <br /> ملاحظة: من الممكن في كثير من الأحيان، خلال المرحلة الأخيرة من هذا الإجراء، لاستخدام الازميل لكسر فتح الخلفي الجمجمة العظام المتبقية من جانب واحد، تماما مثل واحد يفتح الباب. </li></ol><p style=";text-align:right;direction:rtl"><img alt="الشكل 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">الشكل 2: Minipig فتح الجمجمة.</strong> <strong>(A)</strong> التعرض للdorsoposterioص سطح الجمجمة، بما في ذلك إزالة القذالي والعضلات الزمنية. <strong>(B)</strong> إزالة العظم القذالي (CB، المغطاة الجافية المخيخ، OB، عظم القذالي، OC، لقمة القذالي، وSC، والحبل الشوكي). وتستخدم <strong>(C)</strong> ومطرقة وإزميل العظم لاختراق الجمجمة الأمامية ولدخول الجيب الأمامي على مستوى العينين. <strong>(D)</strong> مدى الجيب الأمامي (FS) ويستخدم لإزالة الخارجي سميكة الجمجمة العظام (1)، وتعريض ورقيقة الداخلية الصفيحة العظام (2) التي تغطي المخ. <strong>(E)</strong> إزالة الصفيحة العظام رقيقة، وفضح المخ المغطاة الجافية (السهم). <strong>(F)</strong> وأخيرا، يتم استخدام مطرقة وإزميل العظام للاتصال أفقيا الأمامي والفتحات الخلفية الجمجمة. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> </p><p class="jove_title" style=";text-align:right;direction:rtl"> 4. الدماغ ازالتهاالله </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام ملقط جراحي لرفع الجافية وإنشاء وثيقة شق لطيف على الجيب السهمي العلوي الوريدي باستخدام مشرط جراحي غرامة <strong>(الشكل 3A).</strong> </li><li style=";text-align:right;direction:rtl"> استخدام مقص الجزئي أو الجافية سكين لمواصلة فتح الجافية التي تغطي السطح الظهري من المخ. <br /> ملاحظة: يجب إيلاء عناية خاصة عند إزالة الجافية المقابلة لtentorium المخيخ (الشكل 3B)، والحفاظ على هذا أوراق الجافية سيمنع إزالة الدماغ لاحقة. </li><li style=";text-align:right;direction:rtl"> وضع رأس خنزير عموديا <strong>(الشكل 3C).</strong> </li><li style=";text-align:right;direction:rtl"> استخدام إزميل العظم أو مشرح للافراج عن المخ ventroanterior عن طريق تشريح حادة من البصلة الشمية من الطابق المغطاة الجافية من تجويف الجمجمة <strong>(الشكل 3D).</strong> </li><li style=";text-align:right;direction:rtl"> استخدام مشرط جراحي غرامة لقسم chiasm البصرية يتعرض <strong>(الشكل 3E).</strong> فضح وقسم ساق الغدة النخامية والأعصاب المحرك للعين. </li><li style=";text-align:right;direction:rtl"> الافراج عن الدماغ البطني بواسطة باجتزاء اتحاد نقابات العمالور الأعصاب القحفية <strong>(الشكل 3F)</strong> مع مشرط جراحي على ما يرام. تأكد من أن tentorium المخيخ الجافية وقطعي تماما <strong>(الشكل 3B)،</strong> وهذه ورقة الجافية سيخفض ذلك من خلال الدماغ أثناء عملية الإصدار. </li></ol><p style=";text-align:right;direction:rtl"><img alt="الشكل (3)" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">الشكل 3: إزالة الدماغ Minipig.</strong> <strong>(A)</strong> السحائي فتح مع ملقط الجراحة وسكين الجافية. يجب أن تؤخذ <strong>(B)</strong> العناية إلى شق تماما ورقة الجافية (السهم)، وتقع بين المخ والمخيخ. يتم وضع <strong>(C)</strong> ورأس خنزير عموديا لرؤية أفضل للهياكل قاعدة الجمجمة ومن أجل الجاذبية للمساعدة في تشريد المقصود من الدماغ. <strong>(D)</strong> ومشرح أو إزميل العظم يستخدم لتخفيف البصلة الشمية من قبل قسم حادة من الجافية-covereد قاعدة الجمجمة. واستمر <strong>(E)</strong> وتشريح في اتجاه الخلفي على طول قاعدة الجمجمة عن التعرض وباجتزاء من chiasm البصرية (السهم)، السويقة القمعية، والأعصاب المحرك للعين. اكتمال <strong>(F)</strong> الافراج الدماغ مع قسم الأعصاب القحفية أقل لأنها ابتعدت عن السطح البطني من الدماغ (III، العصب المحرك للعين، IV، العصب البكري، V، العصب مثلث التوائم، والسادس، عصب مبعد). <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> </p><p class="jove_title" style=";text-align:right;direction:rtl"> 5. إزالة الغدة النخامية </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التعرف على ساق الغدة النخامية ورقة محيطها الجافية (والسرج diapragma) في الطابق الجمجمة <strong>الشكل (4A).</strong> </li><li style=";text-align:right;direction:rtl"> شق الوحشي ورقة الجافية إلى ساق الغدة النخامية باستخدام مشرط جراحي غرامة <strong>(الشكل 4B).</strong> </li><li style=";text-align:right;direction:rtl"> استخدام مشرح للافراج عن حفرةuitary ورفعه للخروج من الحفرة النخامية <strong>(الشكل 4C).</strong> </li></ol><p style=";text-align:right;direction:rtl"><img alt="الشكل (4)" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">الشكل 4:</strong> Minipig إزالة الغدة النخامية. تم تحديد <strong>(A)</strong> والحفرة النخامية (*) في الطابق الجمجمة (1، البصلة الشمية، 2، chiasm البصرية، وPF، الحفرة الخلفية في الجمجمة). <strong>(B)</strong> وتغطية الجافية (diagphragm سيلار، (السهم)) هو قطعي أفقيا. <strong>(C)</strong> يتم تحرير النخامية (السهم) مع مشرح ورفعت من الحفرة النخامية. شريط النطاق (AC) = 10 مم. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> </p>

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<p class="jove_content" style=";text-align:right;direction:rtl"> الكتاب ليس لديهم ما يكشف. </p>

<p class="jove_content" style=";text-align:right;direction:rtl"> يتم تنفيذ معظم الدراسات التجريبية علم الأعصاب في أنواع الحيوانات الصغيرة، مثل الفئران والجرذان، حيث يتم تسهيل الوصول إلى الجهاز العصبي المركزي التي كتبها رقيقة skull- والجافية سمك. ومع ذلك، في حيوانات التجارب الكبيرة مثل الخنازير <sup class="xref">1</sup> <sup>و</sup> <sup class="xref">4</sup> <sup>و</sup> <sup class="xref">8</sup> و الأغنام <sup class="xref">32،</sup> والرئيسيات غير البشرية، وسمك كبير من هذه الهياكل يتطلب استخدام أدوات قوية (جدول المواد) ونقاط الدخول المناسبة لإزالة الجمجمة العظمية <strong>(الشكل 2).</strong> مطلوب معرفة تقييد أوراق الجافية <strong>(الشكلان 3</strong> و <strong>4)</strong> قبل أن تتمكن من الوصول إلى الجهاز العصبي المركزي والدماغ إزالة بأمان. </p><p class="jove_content" style=";text-align:right;direction:rtl"> فمن المستحسن أن يترك الجافية سليمة أثناء إزالة الجمجمة العظام، حيث سيؤدي ذلك إلى حماية الدماغ الكامنة من التلف. السابق تثبيت transcardial <sup class="xref">20</sup> بالمثل قد تتصلب وسليghtly يتقلص المخ، مما يسمح العظم وعملية إزالة الجافية التي يتعين القيام بمزيد من السهولة والأمان. وهناك ميزة خاصة للجمجمة خنزير، وعلى النقيض من الغنم والرئيسيات غير البشرية، هو التوسع التدريجي من الجيب الأمامي مع التقدم في السن، والتي قد تكون مفيدة في عملية إزالة الجمجمة العظمية <strong>(الشكل 2).</strong> قد فقا لذلك يتم استخدام تقنية عرض على جميع أنواع الحيوانات الكبيرة، ولكن فقط في الخنازير، خصوصا تلك التي مضى عليها أكثر من 6 أشهر، وسيتم تطوير الجيب الأمامي بما يكفي لتوفير المساعدات في عملية إزالة الجمجمة العظام. وأخيرا، باجتزاء كاملة من الحبل الشوكي أثناء عملية قطع الرأس (الشكل 1B) وباجتزاء الكامل للtentorium المخيخ الجافية <strong>(الشكل 3B)</strong> قبل الافراج الدماغ النهائي مطلوبة للغاية من أجل تجنب الضرر اللاحق إلى الدماغ. </p><p class="jove_content" style=";text-align:right;direction:rtl"> في بعض الدراسات، قد يكون من المفيد أن يكون جزء من الحبل الشوكي العنقي منقاري تعلق على الدماغ.ويمكن الحصول على هذا عن طريق وضع شق قطع الرأس الأولي <strong>(الشكل 1A)</strong> أكثر caudally على الرقبة، مما يتيح الوصول إلى الحبل الشوكي من خلال القرص عنق الرحم فقرات بدلا من مفترق الطرق craniocervical، كما هو موضح في الفيديو الحالي. فإن إزالة الخلفي العظام ثم يجب أن تبدأ من الصفيحة الذيلية المكشوفة. وفضلا عن ذلك، فإن هذه التقنية أن تكون مماثلة، ولذلك فمن المهم أن نتذكر أن الحبل الشوكي يجب مقطوع تماما قبل الانتهاء من قطع الرأس مع التمديد القسري على مستوى القسم <strong>(الشكل 1).</strong> ويتجلى الإجراء الحالي على الحيوانات غير المثبتة، كما استدعت الدراسة الخلفية الفعلية التحليل HPLC من الغدة النخامية مشتقة. ولكن لاحظ أن نفس الأسلوب الدقيق يستخدم على الحيوانات تركز اهتمامها transcardially مع بارافورمالدهيد <sup class="xref"><sup>3،</sup></sup> <sup class="xref"><sup>5،</sup></sup> <sup class="xref"><sup>14،</sup></sup> <sup class="xref"><sup>15،</sup></sup> <sحتى الطبقة = &quot;XREF&quot;&gt; <sup>16،</sup> <sup class="xref"><sup>17،</sup></sup> <sup class="xref"><sup>18،</sup></sup> <sup class="xref"><sup>19،</sup></sup> <sup class="xref"><sup>20،</sup></sup> <sup class="xref"><sup>25،</sup></sup> <sup class="xref"><sup>26،</sup></sup> <sup class="xref"><sup>27،</sup></sup> <sup class="xref"><sup>28،</sup></sup> <sup class="xref">31،</sup> على الرغم من أن جميع الإجراءات في مثل هذه الحالة يجب أن تتم تحت التهوية الجيدة التي يقدمها غطاء الدخان <sup class="xref">20.</sup> اختيار تشريح الأصلي مقابل ولذلك ينبغي <em>في</em> -fixation <em>جملة وتفصيلا</em> وتشريح لاحقة تعتمد فقط على الإجراء اللازم مرحلة ما بعد المعالجة <em>(مثل</em> الأنسجة التقليدية، المناعية، HPLC، وFISH) <sup class="xref">32.</sup> </p><p class="jove_content" style=";text-align:right;direction:rtl"> كما نوقش في الفقرات التالية، ونحن نفضل استخدام الأدوات الجراحية غير الآلية. قد جمجمة الخنازير تتراوح أعمارهم فوق 1-2 سنوات، ومع ذلك، تكون قوية بحيث إزالة الجمجمة مع instr تظاهرuments ليس هو ممكن، مما يستلزم استخدام الأدوات الآلية، مثل craniotomes، مناشير تتأرجح، والتدريبات الكهربائية <sup class="xref">32.</sup> في هذه الحالة، لا يزال المستحسن اتباع الخطوات البروتوكول المشار إليها أعلاه، وبالتالي الاستفادة من تحدث بشكل طبيعي نقاط الدخول الجمجمة والتنمية الجيوب الأمامية. وصول الأمامي قدمت إلى قاعدة الجمجمة أنيقة وسهلة، ولكن يجب إجراء الإفراج حادة وصفت من بصيلات الشم دون توجيه المرئي المباشر <strong>(الشكل 3D)،</strong> وعلى النقيض من اطلاق سراح المزيد الخلفي من الدماغ <strong>(الشكل 3EF).</strong> وبالتالي فإن جزء بطني من بصيلات الشم قد تعاني بعض درجة الضرر غير المنضبط الذي فقط يمكن تجنبها، إذا لزم الأمر، من خلال حفر قاعدة الجمجمة الأمامية من بطني لمبات قبل بدأ عملية الإفراج. لاحظ أن التلاعب، وخصوصا من أنسجة المخ غير المثبتة أثناء إزالة الجمجمة، وقد أشار أن يؤدي إلى histologi<html> كال قطعة أثرية الخلايا العصبية المظلمة، والتي قد تؤدي إلى استنتاجات خاطئة في الدراسات neurotoxicological <sup class="xref">33.</sup> </p><p class="jove_content" style=";text-align:right;direction:rtl"> ويمكن أيضا أن يتم تنفيذ عملية الإزالة الجمجمة العظام مع الآلات مثل craniotomes، مناشير تتأرجح، والتدريبات الكهربائية <sup class="xref">32.</sup> هذه قد تسريع هذه العملية، لكنها لن تزيد أيضا من خطر الأضرار التي لحقت البنى العصبية الكامنة. هذه المعدات يمكن أن يكون مكلفا، وعلى الأرجح أن تكون متوفرة في معظم المختبرات. لذا آثرنا أن تثبت الإجراء الحالي باستخدام المعدات الجراحية غير مزودة بمحركات <strong>(جدول المواد)</strong> التي يسهل الوصول إليها واستخدامها. </p><p class="jove_content" style=";text-align:right;direction:rtl"> وهذا الاسلوب وصف وتوضيح، عندما تستخدم بشكل صحيح، يمكن التعرض وإزالة الدماغ الخنازير بعد الوفاة، الغدة النخامية، و / أو الحبل الشوكي العنقي <strong>(الشكل 5)،</strong> مما أدى إلى قطع الأنسجة التي هي مناسبة تماما لمزيد من التحليل العيانية&quot;XREF&quot;&gt; <sup>5،</sup> <sup class="xref"><sup>6،</sup></sup> <sup class="xref"><sup>19،</sup></sup> <sup class="xref"><sup>26،</sup></sup> <sup class="xref">31،</sup> باجتزاء إلى ألواح الدماغ <sup class="xref">28،</sup> والتحليل الكيميائي لاحقة و / أو معالجة النسيجي <sup class="xref">6</sup> <sup>و</sup> <sup class="xref">14</sup> <sup>و</sup> <sup class="xref">15</sup> <sup>و</sup> <sup class="xref">16</sup> <sup>و</sup> <sup class="xref">17</sup> <sup>و</sup> <sup class="xref">25</sup> <sup>و</sup> <sup class="xref">26</sup> <sup>و</sup> <sup class="xref">27</sup> <sup>و</sup> <sup class="xref">31.</sup> </p>

<html><p class="jove_content" style=";text-align:right;direction:rtl"> أصبحت دراسات علم الأعصاب متعدية الخنازير شعبية متزايدة خلال العقدين الماضيين. حجم كبير من الدماغ الخنازير يمكن استخدام التصوير الدماغ السريرية التقليدية والاستخدام المباشر واختبار إجراءات جراحة الأعصاب والمعدات من عيادة الإنسان <sup class="xref"><sup>1،</sup></sup> <sup class="xref"><sup>2،</sup></sup> <sup class="xref"><sup>3،</sup></sup> <sup class="xref"><sup>4،</sup></sup> <sup class="xref"><sup>5،</sup></sup> <sup class="xref"><sup>6،</sup></sup> <sup class="xref"><sup>7،</sup></sup> <sup class="xref">8.</sup> في السنوات ال 20 الماضية، والخنازير، وخاصة minipigs <em>(على سبيل المثال،</em> غوتنغن minipig)، وقد استخدمت لدراسة طرق العلاج neuromodulatory، مثل زرع الخلايا الجذعية. الفيروسي ترنسفكأيشن النواقل؛ والتحفيز العميق للمخ الموجهة نحو مرض باركنسون، والسمنة، والاكتئاب، ومرض الزهايمر <sup class="xref"><sup>2،</sup></sup> <sup class="xref"><sup>6،</sup></sup>= &quot;XREF&quot;&gt; <sup>9،</sup> <sup class="xref"><sup>10،</sup></sup> <sup class="xref"><sup>11،</sup></sup> <sup class="xref"><sup>12،</sup></sup> <sup class="xref"><sup>13،</sup></sup> <sup class="xref"><sup>14،</sup></sup> <sup class="xref"><sup>15،</sup></sup> <sup class="xref"><sup>16،</sup></sup> <sup class="xref">17.</sup> وقد تبع ذلك وضع نهج التجسيمي والجراحية لمعالجة minipig CNS <sup class="xref">3</sup> <sup>و</sup> <sup class="xref">18</sup> <sup>و</sup> <sup class="xref">19</sup> <sup>و</sup> <sup class="xref">20</sup> <sup>و</sup> <sup class="xref">21.</sup> وقد تم تقييم التغييرات CNS وضعت في الحيوانات الحية باستخدام التصوير الدماغي (PET <sup class="xref">10</sup> <sup>و</sup> <sup class="xref">13</sup> <sup>و</sup> <sup class="xref">22</sup> <sup>و</sup> <sup class="xref">24</sup> و MR <sup class="xref">23)،</sup> قياس المثانة <sup class="xref"><sup>11،</sup></sup> <sup class="xref"><sup>12،</sup></sup> <sup class="xref">25،</sup> تحليل مشية<sup class="xref">17،</sup> وتقييم عصبي <sup class="xref"><sup>9،</sup></sup> <sup class="xref">17،</sup> والفحص بعد الوفاة بناء على الأنسجة وتحليل stereological <sup class="xref"><sup>14،</sup></sup> <sup class="xref"><sup>15،</sup></sup> <sup class="xref"><sup>17،</sup></sup> <sup class="xref"><sup>26،</sup></sup> <sup class="xref"><sup>27،</sup></sup> <sup class="xref">31.</sup> ومع ذلك، وتحليل بعد الوفاة يتطلب التعرض وإزالة الدماغ الخنازير، وهي ليست مهمة سهلة، وسميكة، والجمجمة العظمية والسحائي ليفية تغطي تحيط بالدماغ خنزير. </p><p class="jove_content" style=";text-align:right;direction:rtl"> والهدف من هذه الورقة، والفيديو التعليمي هو شرح طريقة الدماغ الخنازير بعد الوفاة والغدة النخامية قد يتعرض وإزالتها في حالة سليمة في 15-20 دقيقة باستخدام أدوات جراحية غير الآلية. فيديو تعليمي والرسوم التوضيحية التصوير تظهر minipigs الذكور (العمر: 6 شهور، وزن الجسم: 20-25 كغ) تستخدم لدراسة تشريحية على الغدة النخامية minipig. </p>

<table><tbody><tr><td>Heavy Scalpel Handle #4</td><td>FST (Fine Science Tools)</td><td>10008-13</td><td>Good for skin incision and soft tissue removal</td></tr><tr><td>Non-Sterile Scalpel Blades #23</td><td>FST&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Scalpel Handle #7</td><td>FST&amp;nbsp;</td><td>10007-12</td><td>Optimal for dural incision and precision work</td></tr><tr><td>Non-Sterile Scalpel Blades #11</td><td>FST&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>Surgical Forceps</td><td>FST&amp;nbsp;</td><td>11024-18</td><td>The tip of the surgical forceps ensure a firm grip&amp;nbsp;</td></tr><tr><td>Kerrison Bone Punch</td><td>Aesculap Neurosurgery</td><td>FF713R</td><td>Must be robust, bite size 3-5 mm</td></tr><tr><td>Bone Rongeur</td><td>Aesculap Neurosurgery</td><td>MD615</td><td>Must be robust, bite size 15 x 5 mm</td></tr><tr><td>Bone Rongeur</td><td>Aesculap Neurosurgery</td><td>FO551R</td><td>Must be robust, bite size 25 x 15 mm&amp;nbsp;</td></tr><tr><td>Bone Chisel</td><td>Lawton</td><td>67-0335</td><td>The size of the chisel head should not exceed 20 mm</td></tr><tr><td>Mallet (Hammer)</td><td>Millarco</td><td>5624108</td><td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td></tr><tr><td>Micro-Scissor</td><td>FST&amp;nbsp;</td><td>14002-14&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Dissector</td><td>Aesculap Neurosurgery</td><td>OL165R</td><td></td></tr><tr><td>G&amp;ouml;ttingen minipigs</td><td>Ellegaard G&amp;ouml;ttingen Minipigs A/S, Denmark</td><td></td><td></td></tr><tr><td>Euthanimal</td><td></td><td></td><td>pentobarbital</td></tr><tr><td>Ketamine</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Midazolam&amp;nbsp;</td><td>Hameln Pharmaceuticals</td><td></td><td></td></tr></tbody></table>

exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

<p class="jove_content" style=";text-align:right;direction:rtl"> أصبحت الخنازير شعبية متزايدة في والحيوان كبير البحوث متعدية علم الأعصاب كبديل مجدية اقتصاديا وأخلاقيا لالرئيسيات غير البشرية. حجم دماغ كبير من الخنزير يسمح باستخدام التصوير الدماغ السريرية التقليدية والاستخدام المباشر واختبار الإجراءات والمعدات جراحة الأعصاب من العيادة الإنسان. مزيد من التحليل العيانية والنسيجي، ومع ذلك، يتطلب التعرض بعد الوفاة من النظام خنزير العصبي المركزي (CNS) وإزالة المخ لاحقة. هذه ليست مهمة سهلة، والخنزير يتم تغليف CNS من قبل سميكة، والجمجمة العظمية والعمود الفقري. والهدف من هذه الورقة، والفيديو التعليمي هو شرح طريقة لكشف وإزالة الدماغ الخنازير بعد الوفاة والغدة النخامية في حالة سليمة، ومناسبة لتحليل العيانية والنسيجي لاحقة. </p>

<p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"> لمنع المواد الأنسجة من الجفاف، فمن المستحسن لتخزين الدماغ إزالتها والغدة النخامية في جرة مملوءة تثبيتي أو المالحة متساوي التوتر على الفور بعد أن تم إجراء تحليل العيانية. قد يتم تخزين المواد الأنسجة في تثبيتي لسنوات، في حين أن تخزين في المياه المالحة متساوي التوتر، حتى في الثلاجة، سوف تؤدي إلى تسوس الأنسجة مع مرور الوقت. </p><p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"> الغدة النخامية إزالة يمكن أيضا تجميد مباشرة عن طريق الغمر في الأراضي الجافة وتبريد الجليد السائل 2-methylbutane، في حين أن الخنزير الدماغ سليمة كبير جدا لتجميد المباشر <sup class="xref">28.</sup> بدلا من ذلك، فمن المستحسن لشريحة الدماغ الخنازير، كما هو موضح سابقا <sup class="xref">28،</sup> إلى ألواح 9-15 مم موازية الاكليلية الأنسجة التي يمكن تجميد <em>جملة وتفصيلا</em> وناظم البرد-مقطوع إلى 40 أقسام ميكرون سميكة <sup class="xref"><sup>5،</sup></sup> <sup class="xref"><sup>18،</sup></sup> </سوب&gt; <sup class="xref">26</sup> <sup>و</sup> <sup class="xref">28.</sup> بدلا من ذلك، يمكن للمناطق محددة في الدماغ تكون حرة تشريح-من الدماغ سليمة إزالتها أو شرائح لوح الدماغ وتقديمها إلى مزيد من المعالجة النسيجي بعد vibratome باجتزاء <sup class="xref">30،</sup> البارافين / التضمين ميتاكريليت ومشراح باجتزاء <sup class="xref"><sup>6،</sup></sup> <sup class="xref"><sup>17،</sup></sup> <sup class="xref">27،</sup> أو تجميد وناظم البرد باجتزاء <sup class="xref"><sup>6،</sup></sup> <sup class="xref"><sup>14،</sup></sup> <sup class="xref"><sup>15،</sup></sup> <sup class="xref">25.</sup> في وضع لدينا، وضعت جثث الخنازير أخيرا في حاويات بلاستيكية معينة وتخزينها في غرفة التخزين البارد مخصصة حتى يتم جمعها ونقلها إلى مرفق المهينة البيولوجي. </p><p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"> مع استخدام الأدوات الجراحية غير مزودة بمحركات (جدول المواد)، وتقنية وصفها <strong>(الشكلان 1</strong> </stron<html> ز&gt; - <strong>3)</strong> تمكن، في حوالي 15-20 دقيقة، وإزالة الدماغ خنزير سليمة <strong>(الشكل 5AB)،</strong> في حين أن الأعصاب القحفية مقطوعة والغدة النخامية البقاء على اتصال على الأرض الجمجمة <strong>الشكل (4A).</strong> وبالمثل، فإن الغدة النخامية يمكن إزالتها ببساطة، سليمة، بعد إزالة الدماغ المغطي والافراج عن الحجاب الحاجز سرجي الجافية <strong>(الشكل 4</strong> و <strong>5C).</strong> </p><p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"> قد لاحقا أن تقدم الدماغ الناتجة و / أو الغدة النخامية <strong>(الشكل 5)</strong> لتحليل العيانية ذلك، وبصرف النظر عن الفحص البصري المباشر، ويمكن أن تشمل حجم وحجم قياسات <sup class="xref">31.</sup> يمكن أن يتبع ذلك المنحى باجتزاء إلى ألواح الدماغ أصغر <sup class="xref">28</sup> <sup>و</sup> <sup class="xref">29</sup> مناسب للتحليل الكيميائي و / أو مزيد من النسيجي إعداد وتلطيخ، والتحليل المجهري <sup class="xref"><sup>6،</sup></sup>الصورة = &quot;XREF&quot;&gt; <sup>14،</sup> <sup class="xref"><sup>15،</sup></sup> <sup class="xref"><sup>17،</sup></sup> <sup class="xref"><sup>25،</sup></sup> <sup class="xref"><sup>26،</sup></sup> <sup class="xref">27.</sup> </p><p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"><img alt="الرقم 5" src="/files/ftp_upload/55511/55511fig5.jpg" /><br /> <strong>الشكل (5):</strong> <strong> </strong> الدماغ minipig <strong>(A</strong> و <strong>B)</strong> والغدة النخامية <strong>(C).</strong> <strong>(A)</strong> الدماغ، وعرض ظهري جانبي (BS، الدماغ، CB، المخيخ، وCRB، المخ). <strong>(B)</strong> الدماغ، وعرض بطني (BS، الدماغ، CB، المخيخ، وCRB، المخ). <strong>(C)</strong> الغدة النخامية، وعرض الخلفية (AH، النخامية الغدية وNH، النخامية العصبية). شريط النطاق (A و B) = 10 مم، (C) = 5 ملم. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig5large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> </p>

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

<p class="jove_content" style=";text-align:right;direction:rtl"> والهدف من هذه الورقة، والفيديو التعليمي هو شرح طريقة لكشف وإزالة الدماغ الخنازير والغدة النخامية وبعد الوفاة في حالة سليمة، ومناسبة لتحليل العيانية والنسيجي لاحقة. </p>

تعرض الجهاز العصبي المركزي الخنزير للتحليل النسيجي: دليل لقطع الرأس، الجمجمة افتتاح، والدماغ إزالة

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

exposure-pig-cns-for-histological-analysis-manual-for-decapitation

Universidad San Sebastian

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

The Axial Skeleton

SCIENTISTS IN ACTION

15.4K Views.  Aalborg University Hospital.  والهدف من هذه الورقة، والفيديو التعليمي هو شرح طريقة لكشف وإزالة الدماغ الخنازير والغدة النخامية وبعد الوفاة في حالة سليمة، ومناسبة لتحليل العيانية والنسيجي لاحقة. 

Video: Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Cellular Respiration

<h4>Autotrophs and Heterotrophs</h4>

<p>Living organisms require a continuous input of energy to maintain cellular and organismal functions such as growth, repair, movement, defense, and reproduction. Cells can only use chemical energy to fuel their functions, therefore they need to harvest energy from chemical bonds of biomolecules, such as sugars and lipids. Autotrophic organisms, namely plants, algae, and photosynthetic and chemosynthetic bacteria, convert inorganic materials into such biomolecules by harnessing energy from the environment, such as from sunlight during photosynthesis. Heterotrophic organisms are unable to synthesize high-energy biomolecules from inorganic materials, so they obtain energy by consuming carbon compounds produced by other organisms, primarily from autotrophs. When energy is needed, chemical bonds of carbon compounds are broken to harvest the energy stored in these bonds. The processes to harvest energy from biomolecules are called cellular respiration.</p>

<p>Cellular respiration occurs in both autotrophic and heterotrophic organisms, where energy becomes available to the organism most commonly through the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). There are two main types of cellular respiration&#8212;aerobic respiration and anaerobic respiration. Aerobic respiration is a specific type of cellular respiration, in which oxygen (O<sub>2</sub>) is required to create ATP. In this case, glucose (C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>) can be oxidized completely in a series of enzymatic reactions to produce carbon dioxide (CO<sub>2</sub>) and water (H<sub>2</sub>O).</p>

<h4>Aerobic Respiration</h4>

<p>Aerobic respiration occurs in three stages. A process called glycolysis splits glucose into two three-carbon molecules called pyruvate. This process releases energy, some of which is transferred to ATP. Next, pyruvate molecules enter the mitochondria to take part in a series of reactions called the Krebs cycle, also known as the citric acid cycle. This completes the breakdown of glucose, harvesting some of the energy into ATP and transferring electrons onto carrier molecules. In the last stage, known as oxidative phosphorylation, electrons pass through an electron transport system in the mitochondrial inner membrane, which maintains a gradient of hydrogen ions. Cells harness the energy of this proton gradient to generate the majority of the ATP during aerobic respiration.</p>

<h4>Anaerobic Respiration</h4>

<p>Aerobic respiration requires oxygen, however, there are many organisms that live in places where oxygen is not readily available or where other chemicals overwhelm the environment. Extremophiles are bacteria that can live in places such as deep ocean hydrothermal vents or underwater caves. Rather than using oxygen to undergo cellular respiration, these organisms use inorganic acceptors such as nitrate or sulfur, which are more easily obtainable in these harsh environments. This process is called anaerobic respiration.</p>

<p>When oxygen is not present and cellular respiration cannot take place, a special anaerobic respiration called fermentation occurs. Fermentation starts with glycolysis to capture some of the energy stored in glucose into ATP. However, since oxidative phosphorylation does not occur, fermentation produces fewer ATP molecules than aerobic respiration. In humans, fermentation occurs in red blood cells that lack mitochondria, as well in muscles during strenuous activity generating lactic acid as a byproduct, therefore it is named lactic acid fermentation. Some bacteria carry out lactic acid fermentation and are used to make products such as yogurt. In yeast, a process known as alcoholic fermentation generates ethanol and carbon dioxide as byproducts, and has been used by humans to ferment beverages or leaven dough.</p>

<h4>Photosynthesis</h4>

<p>Cellular respiration together with photosynthesis is a feature of the transfer of energy and matter, and highlights the interaction of organisms with their environment and other organisms in the community. Cellular respiration takes place inside individual cells, however, at the scale of ecosystems, the exchange of oxygen and carbon dioxide through photosynthesis and cellular respiration affects atmospheric oxygen and carbon dioxide levels.</p>

<p>Interestingly, the processes of cellular respiration and photosynthesis are directly opposite of one another, where the products of one reaction are the reactants of the other. Photosynthesis produces the glucose that is used in cellular respiration to make ATP. This glucose is then converted back into CO<sub>2</sub> during respiration, which is a reactant used in photosynthesis. More specifically, photosynthesis constructs one glucose molecule from six CO<sub>2</sub> and six H<sub>2</sub>O molecules by capturing energy from sunlight and releases six O<sub>2</sub> molecules as a byproduct. Cellular respiration uses six O<sub>2</sub> molecules to convert one glucose molecule into six CO<sub>2</sub> and six H<sub>2</sub>O molecules while harnessing energy as ATP and heat.</p>

<h4>Measuring Respiration</h4>

<p>Scientists can measure the rate of cellular respiration using a respirometer by assessing the rate of exchange of oxygen. Understanding the Ideal Gas Law is of fundamental importance for knowing how the respirometer functions. The Ideal Gas Law states that the number of gas molecules in a container can be determined from the pressure, volume, and temperature. More specifically, the product of the volume and pressure of a gas equals the product of the number of gas molecules, the ideal gas constant and the temperature of the gas. Respirometers contain potassium hydroxide which traps carbon dioxide that is produced by respiration in solid form as potassium carbonate. When cells consume oxygen, the gas volume in the respirometer system decreases with no carbon dioxide to increase it back up, allowing scientists to calculate the amount of oxygen used using the ideal gas equation.</p>

<p>Cellular respiration is an important process that creates usable energy for organisms, therefore, studying the contexts in which it is improved or impeded is not only interesting, but also necessary. Especially, mitochondria are essential for cellular respiration and any conditions that affect mitochondrial health have immense consequences for the health of the organism. For instance, mitochondrial myopathies are a group of neuromuscular diseases which are caused by mitochondrial damage, affecting predominantly nerve and muscle cells, which require high levels of energy to function<sup>1</sup>. Moreover, many poisons work by inhibiting cellular respiration. For example, cyanide inhibits the production of ATP through oxidative phosphorylation, thus understanding the mechanisms cyanide or other metabolic poisons enables treatment of individuals who have been exposed to them<sup>2</sup>. Similarly, some medications such as certain antibiotics, chemotherapeutics, statins, and anesthetics can also interfere with mitochondrial function and may not be suitable to treat patients that have mitochondrial disorders<sup>3</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Lin, MT and Beal, MF.</strong> Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. <em>Nature. </em>2006, Vol. 443, (787-95).</li>
	<li><strong>Beasley, DMG and Glass, WI.</strong> Cyanide poisoning: pathophysiology and treatment recommendations. <em>Occupational Medicine. </em>Vol. 48, 7 (427-31).</li>
	<li><strong>Finsterer, J and Segall, L.</strong> Drugs interfering with mitochondrial disorders. <em>Drug Chem Toxicol. </em>2010 , Vol. 33, 2 (138-51).</li>
</ol>


Cellular Respiration: Quantification using Microrespirometers - Concept

التنفس الخلوي

Autotrophs and Heterotrophs

Living organisms require a continuous input of energy to maintain cellular and organismal functions such as growth, repair, ...

JoVE Lab Manual

Biology

Cellular Processes

Cell Structure

<h4>Background</h4>
<p>Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19<sup>th</sup> century, the Cell Theory was proposed to define a cell, which states:</p>
<ul>
	<li>Every living organism is made up of one or more cells.</li>
	<li>The cells are the functional units of all organisms.</li>
	<li>All cells arise from preexisting cells.</li>
</ul>
<p>All cells share common features such as having a plasma membrane, a cytoplasm, DNA, and ribosomes. A plasma membrane is a phospholipid bilayer that surrounds the cell. This thin and fluid layer around the cells serves to isolate the cell&#8217;s contents from its environment and regulates the material exchange with its environment, while also facilitating interactions with other cells. Inside the plasma membrane, the cell is filled with a gel-like fluid called cytoplasm that contains organic molecules, salts, and other materials that are vital for the cell&#8217;s functions. Therefore, biochemical reactions that support life, which are known as metabolic processes, take place inside the cytoplasm. The types of metabolic processes that a cell can execute depend on its genetic information. All cells use DNA as the genetic material, which is the hereditary blueprint to construct cellular structures and products. Finally, all cells use ribosomes to synthesize their protein products.</p>
<p>There are two types of cells based on their genetic material location: prokaryotic, which means &#8220;before nucleus&#8221;, and eukaryotic, which means &#8220;true nucleus&#8221;. Therefore, while both types of organisms have DNA, prokaryotes like bacteria have nucleoids, or &#8220;nucleus-like&#8221; components instead of a nucleus, whereas eukaryotes possess true, membrane-bound nuclei to contain their DNA. Moreover, prokaryotes are relatively small, around 0.1&#8211;5.0 micrometers (&#181;m), in comparison to eukaryotes that can typically range from 10-100 &#181;m in size. The small size of prokaryotes allows quick and effortless distribution of materials within the cell and execution of metabolic processes, as well as swift removal of waste or other products from the cell. Hence, eukaryotic cells possess specialized structures known as organelles, such as mitochondria or Golgi apparatus, to enable the execution of vital functions.</p>
<h4>The Eukaryotic Cell</h4>
<p>The eukaryotic cell is a shared derived trait of all eukaryotes, meaning that it had a single origin that has since been inherited by all eukaryotes. The earliest eukaryotic cells are seen in fossils about 2.4 billion years ago, and are recognizable because they are larger than prokaryotic cells<sup>1</sup>. The origin of this cell type resulted from an endosymbiotic event in which one amoeba-like cell engulfed a micrococcal bacteria and formed a stable coexistence<sup>2</sup>. The engulfed bacteria evolved into the first energy-producing organelles, mitochondria, which are the aerobic metabolism organelles in the cell. Mitochondria have their own separate genome and are similar in size to prokaryotes. They contain two layers of membranes that enclose two distinct compartments. Some of the reactions that break down high-energy biomolecules occur in the inner compartment, whereas the outer compartment houses the reactions that capture the energy released from these compounds into adenosine triphosphate (ATP) molecules to be used as the energy currency of the cell.</p>
<p>Nuclei and mitochondria are not the only shared structures of eukaryotic cells. Other ubiquitous eukaryotic organelles are the smooth and rough endoplasmic reticulum (ER), Golgi apparatus, lysosomes and vacuoles. Endoplasmic reticulum simply means &#8220;network inside the plasma&#8221; and as its name indicates it is a large network of membranes inside the cell, especially around the nucleus. Parts of the rough ER extend from the nuclear membrane and are distinguished from the smooth ER by their rough appearance due to numerous ribosomes on their surface. Rough ER is the site for protein synthesis, such as the proteins that are embedded in the plasma membrane or the proteins that are secreted from the cell. In contrast, smooth ER produces lipid-based products but also contains enzymes to detoxify harmful chemicals. Hence liver cells contain abundant smooth ER. Also, muscle cells contain significant amounts of smooth ER because of the calcium-storing function of this organelle, which is essential for muscle contraction. The Golgi apparatus sorts, modifies, and packages cellular products inside vesicles, which fuse with the plasma membrane to release the products. Some of the proteins that are produced in the rough ER are intracellular digestive enzymes. These enzymes are packed in the Golgi apparatus into special vesicles called lysosomes. The major function of the lysosomes is to digest the food particles engulfed by the cell as well as old cell parts. Vacuoles are sacs of cell membrane that serve as storage units within cells. They may serve to store water to regulate the cell&#8217;s water content as well as store metabolic products or even poisonous molecules, depending on the cell type and the organism.</p>
<h4>Kingdom-specific Organelles</h4>
<p>Eukaryotic cells also developed distinct organelles, specific for each kingdom. For instance, kingdom Plantae and Animalia are both eukaryotic, however the organelles of plant and animal cells differ in key ways that allow them to carry out their lives as producers and consumers, respectively. Terrestrial plants need to grow tall and have rigid stems to hold leaves, which they use for photosynthesis. They also need to be able to retain water taken up by roots. Their cells reflect these specific needs. Unlike animal cells, plant cells have chloroplasts, which are used for photosynthesis and often contain the green pigment chlorophyll. Additionally, they are surrounded by cell walls, which are rigid outer layers made of cellulose to support growth and water retention. Because they need to store large amounts of water to maintain the water pressure in the cell, they have larger vacuoles than animal cells. In addition, plant cells also have another type of specialized storage organelles called plastids, which contain pigments as well as photosynthetic products such as starch. These differences are noticeable and distinguish plant cells from animal cells: plant cells generally have a regular, rectangular shape due to their rigid cell walls, while animal cells are rounded and more irregular.</p>
<h4>Microscopy</h4>
<p>Some cells, such as frog oocytes, are large enough to be seen with the naked eye, yet most cells cannot be seen without any visual aid. Therefore, scientists use microscopy techniques to study cell structures and distinguish cell types from one another. While microscopes are able to magnify objects that are difficult or impossible to be see with the human eye, most tissues naturally lack pigmentation. Therefore, solutions have been created that can selectively stain cells based on their molecular composition. This allows researchers to distinguish between organelles in a cell, tissue types in a plant stem, and fat layers in animals, just to name a few examples. Methylene blue dye stains nucleic acids of dead cells, binding to negatively charged DNA. Safranin solution is another biological dye that stains cell nuclei red. Cells only need to be in the staining solutions for a short period of time, and can be mounted immediately following the staining step. The commonly used mounting techniques are wet mount and oil immersion. A wet mount is created by collecting a sample and putting it on a glass slide with liquid between the slide and the coverslip. Cell samples are suspended in liquids like water or glycerol. Glycerol is better to use with live cultures, because it prevents the proliferation of bacteria<sup>3</sup>. Immersion oil can be added on top of the coverslip to improve viewing of the sample at high magnification. This is accomplished because the oil has the same refractive index as glass, which means that it allows light to pass through it as well as glass would. The glass-air interface scatters light more than oil or glass, so the clarity of the image is affected when samples are mounted &#8220;dry&#8221;, or without oil. Once the cells are stained and mounted, they are ready to be studied under the microscope.</p>
<p>There are various microscopy techniques from electron scanning technology that has allowed researchers to view objects on an atomic level to fluorescent live cell imaging that allows real-time monitoring of the movement of molecules within individual cells<sup>4</sup>. Bright-field microscopy is the simplest microscopy technique, requiring only a halogen source light, a condenser lens to focus the light, an ocular lens to view the image, and an objective lens to magnify the image. With any microscopy technique, it is important to understand the parts of a microscope before using one. In general, compound microscopes used for bright-field imaging have an eyepiece at the top of the scope, which is attached to the head and objectives. The eyepiece has a magnification of 10X, and objective lenses are set to a particular magnification in a range of 4X-100X. There are between three and five objectives on a standard microscope. The objectives point down to the stage, which is where a specimen is placed for viewing. The stage often has mechanical parts and stage clips to hold a slide and move it around while viewing. An aperture is a hole in the stage for light to pass through. This light is controlled by an adjustable condenser lens above an illuminator, or light source. To control the zoom of the stage for the object being viewed, microscopes feature coarse and fine focus adjustment knobs. The coarse focus knob moves on a larger scale than the fine focus, but they are on the same axis. The fine focus is useful when the object on the stage has been brought close to the objectives. It is important not to let the objective lens touch the object on the stage, as it can scratch the lens. Objects should always be first viewed on the lowest magnification objective and clearly focused before switching to higher magnification objectives.</p>
<p>Microscopy is an important tool for many aspects of the medical field including research, diagnosis, and treatment. This has the application of using nanotechnology in medicine, as a new treatment method in place of a more invasive surgery<sup>5</sup>. Surgeons also make use of microscopes, some of which have been modified to be mounted on a surgeon&#8217;s head and are operated with foot pedals. These are of much lower magnification than even the light microscopes used today, but they facilitate the safe execution of delicate procedures, such as optical and neurosurgery.</p>
<h4>References</h4>
<ol class="references">
	<li><strong>Bengtson S, Rasmussen B, Ivarsson M, Muhling J, Broman C, Marone F, Stampanoni M, Bekker A.</strong> Fungus-like mycelial fossils in 2.4-billion-year-old vesicular basalt. <em>Nature Ecology &#38; Evolution. </em>2017, Vol. 1, Article number: 0141.</li>
	<li><strong>Vellai T, Vida G.</strong> The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. <em>Proc. R. Soc. Lond. B. </em>1999, Vol. 266, 1571-1577.</li>
	<li><strong>Gouet V, Roger G, Fonty C, Andre P.</strong> Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi. <em>Current Microbiology. </em>24, 1992, Vol. 4, 197-201.</li>
	<li><strong>Cognet L, Leduc C, Lounis B.</strong> Advances in live-cell single-particle tracking and dynamic super-resolution imaging. <em>Curr Opin Chem Biol. </em>2014, Jun; 20:78-85.</li>
	<li><strong>Asiyanbola B, Soboyejo W.</strong> For the surgeon: an introduction to nanotechnology. <em>J Surg Educ. </em>2008, Vol. 65, 2 (155-61).</li>
</ol>

Cell Structure: Visualizing Onion and Human Cells - Concept

هيكل الخلية

Background
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, ...

Fundamentals

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

إستخلاص

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

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Chemistry

Lab Manual Sub Articles

Lab Techniques

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Determining the Density of an Egg</strong><button class="expand">Expand</button>
	<p>Density is a measure of how compact a substance or object is, and it is calculated as mass divided by volume. When a dense object is placed in a liquid with a lower density, the object sinks; when the object is less dense than the liquid, it floats. When the densities are the same, the object will be suspended in the liquid. In this experiment, we&#39;ll determine the density of an egg by creating a solution with the same density. We&#39;ll measure the density of the salt solution using different volume measurement glassware and determine which is the most accurate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-1">
		<div class="lab-step">To begin, wear the appropriate personal protective equipment, including close-toed shoes, long pants, long-sleeved shirt, lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-2">
		<div class="lab-step">Measure the mass of the egg. Set a weigh boat on the balance and tare it. Carefully set the egg in the weigh boat while you record the mass in your notebook.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare the salt solution that will be used to suspend your egg. Use the 600-mL beaker and fill it 3/4 of the way full with deionized water.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Gently place your egg in the beaker, and then add about one teaspoon of NaCl into the water. Stir the solution gently until the NaCl dissolves. Continue to add NaCl until the egg floats just below the surface.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use different glassware to measure the volume of the salt solution. Weigh the 50-mL graduated cylinder on the top loading balance and record the mass. Then, measure 20 mL of your salt solution and record the volume in your notebook. <strong>Note: </strong>Place a blank sheet of paper behind the graduated cylinder to read the volume more clearly. Always view the measurement at eye level and measure the volume from the bottom of the meniscus. The lines on the graduated cylinder are 0.2 mL apart, meaning that you can read the volume to the hundredths place.
		<h4><b>Table 1: Measuring Mass</b></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>Container</strong></td>
					<td style="text-align: center;"><strong>Mass of empty container (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of full containter (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of solution (g)</strong></td>
					<td style="text-align: center;"><strong>Percent error</strong></td>
				</tr>
				<tr>
					<td>50-mL graduated cylinder</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>10-mL volumetric pipette</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>50-mL beaker</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>20-mL volumetric flask</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/41/Table_1._Measuring_Mass.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Weigh the filled graduated cylinder and record the mass in your notebook. Then, pour the salt solution back into the 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use the volumetric pipette to measure volume. First, weigh the empty 50-mL beaker and record the mass in your notebook. Then, carefully measure 10 mL of the salt solution using the volumetric pipette and dispense the salt solution into the beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure another 10 mL and add it to the beaker. Weigh the filled beaker and record the mass in your notebook. Then, empty, rinse, and dry the beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use the volumetric flask to measure volume. Weigh the empty 20-mL volumetric flask and record the mass. Then, carefully fill the flask with the salt solution to just below the 20-mL fill mark. Do not fill it all the way. Use an eyedropper to finish filling the flask so that the bottom of the meniscus just touches the fill line on the neck of the flask. Then, weigh the full flask and record the weight.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Lastly, use the 50-mL beaker to measure volume. Weigh the empty beaker, then fill the beaker with salt solution to the 20-mL line and weigh the full beaker. Record the masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure the displacement of the egg when it is submerged in the salt solution. First, remove the egg and look at the level of the salt solution in the 600-mL beaker. <strong>Note:</strong> If it is not at a marked line, add deionized water to the solution so that the meniscus is at a readable volume and record the volume.&#160;
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Measurements</strong></td>
					<td><strong>Salt solution using 20-mL volumetric flask&#160;</strong></td>
					<td><strong>Egg</strong></td>
				</tr>
				<tr>
					<td>Mass (g)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Displacement of water (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Inner diameter of 600-mL beaker (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (cm&#179;)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (mL)</td>
					<td style="text-align: center;">20</td>
					<td></td>
				</tr>
				<tr>
					<td>Density (g/mL)</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<strong><a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/41/Table_2._Measuring_Density.xlsx" target="_blank">Click Here to download Table 2</a> </strong></div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Gently place the egg in the beaker.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Use a ruler to measure the new height of the solution level and record the height. Then, place the ruler across the top of the beaker to measure the inner diameter.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Calculate the new volume from these measurements. Subtract the initial volume to obtain the displacement of the egg (volume).</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">To clean up from the experiment, return the egg to your instructor and pour the salt solution down the sink. Rinse all used glassware with tap water and set them out to dry. Finally, return the sodium chloride to your instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Calculate the mass of the solution in each of the different volumetric glassware by subtracting the empty glassware mass from the full glassware mass.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Keep track of uncertainty in your measurement by using significant figures. For example, when we look at the graduated cylinder measurement, the weight of the empty container is 90.92, and the weight of the full container is 111.59. The weight measurements have significant figures to the hundredths place, so it is important that significant figures are maintained throughout our calculations. Thus, the mass of the 20-mL salt solution is 20.67 g.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The 50-mL beaker weighed 28.65 g when it was empty and 47.96 g when it contained 20 mL of salt solution. Therefore, the mass of 20 mL of the salt solution is 19.31 g.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Repeat the calculation to obtain the mass of 20 mL of salt solution that was measured using the 10-mL pipette and the 200-mL volumetric flask.<b> Note:</b> Generally speaking, volumetric measurements are more accurate when using a volumetric flask.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Look at the accuracy of the other methods compared to the volumetric flask in terms of percent error. Percent error is calculated by subtracting the theoretical value from the experimental value, then dividing by the theoretical value times 100.
		<ul>
			<li><img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/41/41_Procedure_1b.jpg" /></li>
		</ul>
		</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the volumetric flask measurement as the theoretical value to determine the percent error for the other volume measurement methods. As expected, the volume measurement that uses the markings on the beaker is the least accurate.<strong> Note: </strong>We also know this because the known density of pure water is 1 g/mL. Thus, 20 mL of pure water weighs 20 g. The density of the salt solution must be higher than 20 g since salt was added to the water.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the density of the salt solution using the volumetric flask measurement. Divide the mass of the solution by the volume of the solution. Thus, the density of the salt solution is 1.053 g/mL.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the calculated density of the salt solution to the density of the egg. The volume calculated using the displacement of water in a beaker was found to be 61.14 cm<sup>3</sup> or 61.14 mL. The weight of the egg was measured to be 60.15 g, so its density is 0.9838 g/mL. The disparity between the density of the egg and the density of the salt solution arises from error introduced in both calculations, primarily due to volume measurement readings.</div>
		</li>
	</ul>
	</li>
</ol>

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

تقنيات المختبر

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

	Determining the Density of an EggExpand
	Density is a measure of how compact a substance or ...

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

انقراض

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

Ecology

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</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">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

التنوع الميكروبي والفطري

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Evolution

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing the Cell Cycle in a Root Tip
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the slice of onion into a microcentrifuge tube labeled &#8220;N&#8221; for the nocodazole condition. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled &#8220;C&#8221; for the control condition.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Add 2 &#181;L of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When incubation is complete, set a heat block to 60 &#186;C.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Wash the nocodazole treated tips with water and then aspirate the liquid.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Repeat this wash two more times, for a total of three washes. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 &#186;C heat block for 12 - 15 min, then discard the acid into a waste flask.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then place 50 &#181;L of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, preform three distilled water washes as in step 10. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Label one microscope slide as 'Control' and another as 'Nocodazole'. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Understanding the Loss of Cell Cycle Control
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. 
</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">Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/10/Table_1_Cell_division.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">If there are any cells undergoing mitosis, note what stages of cell division you see.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the percentage of cells in each stage of mitosis in Table 1. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Procedure

انقسام الخلية

Observing the Cell Cycle in a Root Tip
ExpandHypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a ...

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

سلوك

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 ...

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To prepare for the experiment, be sure to obtain cultures of <em>Drosophila melanogaster</em> at least two weeks ahead of the planned experiment day. NOTE: Because adult females store sperm after mating, you will need this time to generate virgin females by breeding the flies.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">You should get two stock cultures: wildtype, that is homozygous dominant red eyed flies, and white chromosome one mutant, which are homozygous recessive white eyed flies.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Purchase or create <em>Drosophila </em>habitats consisting of a clean vial, 3 cm of prepared food media, and a stopper to close the open end of the vial.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">When not being moved or counted, keep the flies in an incubator or room kept at 23 - 25 &#176;C, where the lifecycle will take around 12 days. NOTE: From the egg, <em>Drosophila </em>progress through four main stages of development: embryo, larva, pupa, and adult. The complete lifecycle takes about 10 days at 25 &#176;C, but it can be affected by temperature. For example, at 18 &#176;C, the lifecycle is about 19 days and at 29 &#176;C, the lifecycle is only seven days.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">As an overview, to set up for the student experiment, you will generate vials for each student group containing white eyed virgin female flies only, white eyed males only, red eyed virgin females only, and red eyed males only. These will be referred to as the experimental vials.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When your vials from the supplier arrive, referred to as the stock vials, these will contain a mix of male and female flies, as well as larvae, pupae, and eggs.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Beginning with your stock vials, you will take around 3 - 5 males and the same number of females, keeping white eyed and red eyed flies separate at all times, and place these together into a new nursery vial containing food. NOTE: You can sex the flies using a magnifying glass. Look at the posterior of the fly. Males are slightly smaller and have darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">You will leave the flies in these nursery vials for around 3 - 5 days to mate and lay eggs.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">After this, you will move them back to their stock vials.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, you will observe as the eggs and larvae in the nursery vials mature, checking the vials every 6 - 8 hours. As it takes females around 8 - 10 hours to become sexually mature, this should ensure that any females found in the nursery vials are virgin.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">As the new adult flies appear, you should transfer them from the nursery to the appropriate experimental tube. For example, if it is a male, transfer it to the experimental male tube for the appropriate eye color. If it is a female, it should be placed in an experimental vial labeled with eye color and virgin female.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Keep adding flies to the appropriate vials until each experimental vial has at least five flies. NOTE: You will need to make enough experimental vials so that each student group has one of each eye color type and sex, with a few class spares. You should create enough nursery vials to account for this. A good rule of thumb is that it generally takes about two weeks for 25 healthy adults to produce about 50 new adults.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To begin the process, first label empty habitats to make the nursery vials to raise larvae and pupae for each strain of flies: red eyed and white eyed.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">To move the adult flies into the nursery vial to lay eggs, use an anesthesia wand according to the manufacturer&#39;s instructions, placing it into the stock vial of mature flies. Wait until the animals become unconscious. Now they can be gently tapped onto a piece of white paper and sorted into piles of the same sex using a brush.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Transfer a few male and female flies to the appropriate vial labeled with their eye color phenotype and sex.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Place the nursery vials to incubate at 23 to 25 &#176;C and the flies will lay eggs.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Remove the adults after 3 - 4 days using anesthesia and put them back into the stock vial.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Remove any adult flies that have eclosed in the 6 - 8 hour period using an anesthesia wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, tip out the adults onto paper and sort them into groups based on their sex before placing each fly into an appropriate experimental vial marked male or virgin female and red or white for the phenotype. NOTE: Each experimental vial should contain five flies. Once this number is reached, then a new vial should be started. If you miss the 6 - 8 hour window, females may not be virgins, so these should be discarded into a morgue tube containing 70% ethanol or returned to the stock vial. Males do not need to be virgins, but do need to be kept in male only containers.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Each lab group will need one tube of five virgin red eyed females, one of five virgin white eyed females, one tube of red eyed males, and one of five white eyed males. In addition, they will need a morgue vial containing ethanol, two anesthesia wands, fly anesthetic, a paintbrush, a magnifying glass, some plain paper, and two empty fly vials for crosses.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Prep

علم الوراثة للكائنات الحية

REF and WEF Fly Genetics
ExpandTo prepare for the experiment, be sure to obtain cultures of Drosophila melanogaster at least two weeks ahead of the ...

Genetics

Animal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Form and Function of Crickets and Crayfish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: To observe the relationship between form and function in animals, you will be examining and dissecting a cricket and a crayfish. Each pair of students will receive one of each of these organisms. When dissecting, be sure to wear gloves, closed-toed shoes, and safety goggles. Take care when handling dissecting tools, as they may be sharp. HYPOTHESES: In this experiment, the experimental hypothesis could be that the cricket and crayfish have similar body structures because they are both arthropods, but that they will have some specific adaptations for their different habitats and lifestyles. The null hypothesis might be that the cricket and crayfish have identical adaptations and body plans.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, place the cricket on its back and observe its appendages.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">In Table 1, make detailed notes about your observations and try to predict the potential function of each of the forms you observe. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_1_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Do the same with the wings of the cricket if they are present.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Consider how the structure of the wings benefits the cricket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If the cricket does not have wings, consider the reasons behind their absence. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If the cricket is female, it will have an ovipositor. This can be observed as a long dark tube protruding from the posterior end and it is used to deposit eggs.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Consider why the ovipositor is useful. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Next, flip the cricket over onto its back and observe the mouth parts under a microscope. NOTE: Use a probe to move structures when necessary.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Make a hypothesis about the size and type of prey the cricket consumes based upon your observations.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Continue to examine the external parts of the cricket, making hypotheses about their function until you have filled at least five rows in the table with your observations.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using tweezers to hold the cricket down, carefully remove the wings and legs from the body with the scissors.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, position the body of the cricket so that its underside is facing down.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Again with the scissors, cut the thin outer exoskeleton from the anus to the head trying not to damage any of the organs underneath.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Use the tweezers to open the incision and dissecting pins to hold open the exoskeleton.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Next, use the water pitcher to carefully add water to the Petri dish until it reaches just below the top edge. This should make the exposed organs of the cricket easier to see.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Move the Petri dish to the stage of the dissecting microscope and turn on the light.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Use the cricket figure as a reference and observe the organs under the dissecting microscope. Where necessary, use the probe to move organs that may be blocking others.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Identify as many internal structures as possible and fill out the form and predicted function for at least three internal structures.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you&#39;ll observe the outer and inner structures of the crayfish. Begin with the crayfish on its back and observe the appendages.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">In Table 1, take notes about the structures you notice.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Observe the tail. Devise a hypothesis for its jointed structure. Consider how a crayfish moves in its environment.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, with the crayfish still on its back, look for the swimmerets under its tail.  NOTE: If the crayfish is male, the swimmerets nearest the legs are long extending between the legs. These are specialized reproductive structures that transfer sperm to the female. Females do not have these long structures, but instead have swimmerets that store developing embryos.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Consider the benefits of this strategy. Be sure to observe both a male and female crayfish, checking with neighboring students to identify these structures.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Next, observe the mouth parts of your crayfish using a probe.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Based on these structures, make a hypothesis about the size and type of prey you think crayfish consume.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Before beginning the dissection, be sure to fill in Table 1 with at least five external structures you observed in the crayfish along with their predicted function. With the organism lying legs down, use scissors to cut an incision in the exoskeleton from the tail to between the eyes. Take care to not disturb the organs underneath.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Next, remove the carapace as demonstrated in the video.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Open the tail and use the pins to secure the exoskeleton down to the dissecting tray exposing the organs.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Observe the organs first with the naked eye. Use the figure to identify the different body parts of the animal.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Then, if there is enough space on the stage, place the dissecting tray with the crayfish under the dissecting microscope to view these structures more closely.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">Fill out at least three rows of Table 1 with these observations and predicted functions of internal structures in the crayfish.
</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, first separate into groups. Within your groups, compare observations from your tables.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these observations, create class predictions that relate the observed forms to the hypothesized functions for both the cricket and the crayfish. As a group, fill out Table 2 with at least five of these predictions and discuss how you would go about testing these hypotheses. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_2_Animal_Diversity.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 the internal and external structures you observed in the cricket and crayfish. Fill out Table 3 with similarities and differences you noticed in the structures of these organisms. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_3_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
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