Name: a procedure to observe context-induced renewal of pavlovian-conditioned alcohol-seeking behavior in rats
Uploaded: 2014-09-19T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats at JoVE.com

<p class='jove_content'>This work was supported by the National Institute on Alcohol Abuse and Alcoholism (RO1 AA014925 awarded to Dr. Patricia H. Janak at University of California, San Francisco, CA, USA), Fonds de recherche du Quebec &#8211; Sant&#233; (NC) and the Natural Sciences and Engineering Research Council (NC, 387224-2010). The open-access publication of this article was made possible by sponsorship from Med Associates Inc. </p>

<p class="jove_content"> Todos os procedimentos foram aprovados pelo Comitê de Ética em Pesquisa Animal da Universidade de Concordia e concordar com as recomendações do Canadian Council on Animal Care. </p><p class="jove_title"> 1. Os animais </p><ol><li> Obter ratos machos Long-Evans (220-240 g à chegada). </li><li> Manter ratos em uma temperatura (21 ° C) e umidade (44%), controlada biotério, em um ciclo claro-escuro de 12 horas com as luzes acesas às 7:00 am. </li><li> Ratos da casa-Pair na chegada. Após 3 dias, os ratos da casa individualmente em gaiolas caixa de sapatos de plástico com cama beta-chip, um osso de nylon, e acesso livre à ração padrão e água. Use tampas gaiola que tem ilhós duplas para permitir a posterior colocação de uma segunda garrafa, para além da garrafa de água. NOTA: Os ratos podem ser alojados individualmente, assim que chegar na unidade de cuidados animais. </li><li> Comece a lidar com os ratos diariamente. NOTA: Como um guia, uma semana de manipulação é geralmente suficiente para se aclimatar-los a ser picked acima e tratadas pelo experimentador. </li></ol><p class="jove_title"> 2. intermitente Etanol Acesso no Lar da gaiola </p><p class="jove_content"> NOTA: Conduta acesso etanol intermitente na gaiola casa para garantir que os ratos vão beber quantidades fisiologicamente relevantes de etanol antes da fase de treinamento comportamental do experimento começa <sup>17-19.</sup> Iniciar este procedimento durante a fase de luz, embora possa ser iniciada em qualquer momento durante o ciclo de luz ou escuro. </p><ol><li> Prepare duas garrafas para cada rato. <ol><li> Use um 100 ml de plástico cilindro graduado como a garrafa de álcool e uma garrafa de água de plástico padrão de 473 ml. Use uma pequena garrafa de etanol de capacidade cheia quase a capacidade máxima de volume, para minimizar a perda de etanol devido ao vazamento e evaporação. </li><li> Encher um frasco com etanol a 15%, feito por diluição de etanol a 95% em água da torneira. NOTA: Outras concentrações de etanol, tal como 10% ou 20%, pode ser utilizado em lugar de 15% de etanol. </li><li> Fill a segunda garrafa com água da torneira. </li><li> Coloque uma tampa de borracha, com um tubo sipper que contém um rolamento de esferas para minimizar as fugas, em cada frasco. </li></ol></li><li> Configure duas gaiolas de controle, tratados de forma idêntica às gaiolas com a ressalva de que as gaiolas de controle não contêm ratos ou roupas de cama. <ol><li> Prepare etanol e garrafas de água para as gaiolas de controle, conforme especificado nas etapas 2.1.1 - 2.1.4. </li><li> Utilizar as medidas obtidas das gaiolas de controlo para controlar o derramamento e / ou evaporação que pode ocorrer ao longo da sessão de acesso etanol (conforme descrito nos passos 2.8.1 - 2.8.2). </li></ol></li><li> Pesar todos os frascos de etanol e água. Anote todos os pesos. </li><li> Pesar todos os ratos. Anote todos os pesos. </li><li> Inserir o frasco de etanol e a garrafa de água em cada gaiola e controlar gaiola ao mesmo tempo. Deixe as duas garrafas nas gaiolas para 24 hr. </li><li> No final do período de 24 horas, remover ambas as garrafas dagaiolas ao mesmo tempo. </li><li> Pesar todos os frascos de etanol e água. Anote todos os pesos. </li><li> Para determinar a quantidade de etanol e água foi consumida durante o período de 24 horas, subtrair o peso de cada frasco em que foi retirado da gaiola de origem a partir do peso da garrafa, quando foi colocado na gaiola 24 horas antes de cada rato. <ol><li> Faça o mesmo para as duas gaiolas de controle, e depois calcular a quantidade média de derrame para cada fluido. </li><li> Subtrair o valor médio do etanol e derramamento de água, respectivamente, a partir das medidas de etanol e consumo de água correspondentes para cada rato. </li><li> Use esses derrames controlado diferença medidas para calcular o consumo de etanol em g / kg, o consumo de água, e de preferência etanol. </li></ol></li><li> Para calcular o consumo de etanol (g / kg), primeiro converter o peso (g) de uma solução de etanol consumido em volume (ml) com base na densidade da solução total, (componentes de etanol + água), de acordopara o volume de fórmula (ml) = peso (g) / densidade (g / ml). NOTA: A densidade da solução irá variar como uma função da concentração de etanol. <ol><li> Em seguida, multiplicar o volume (ml) de uma solução de etanol consumida pela densidade (g / ml) de etanol escalado para a solução percentagem específica utilizada, para se obter o peso (g) de álcool consumido. </li><li> Dividir o peso (g) de álcool consumido pelo peso do rato (kg) para se obter o g / kg medida de consumo de etanol. </li></ol></li><li> Para calcular a preferência etanol, expressar a ingestão de etanol (ml) como uma percentagem do total de fluido (etanol + água, mL) de admissão. </li><li> Retorne a garrafa de água só volta a gaiola do rato. Para as próximas 24 horas, dar o acesso à água ratos, mas não o etanol. Em vez de uma garrafa de etanol, colocar uma segunda garrafa de água pré-pesado na gaiola de alojamento. </li><li> No final do período de 24 horas, remover ambas as garrafas de água a partir da gaiola ao mesmo tempo. </li><li> Pesar os dois conjuntos de garrafas de água. Grave tudo weights. </li><li> Subtrair o peso de cada frasco em que foi retirado da gaiola de origem a partir do peso da garrafa, quando foi colocado na gaiola 24 horas mais cedo, subtraindo a média da medida de derramamento obtida a partir de gaiolas de controlo tal como descrito nos passos 2.8.1 - 2.8.2. </li><li> Use essas diferenças medidas controlado por derrames para calcular o consumo de água. </li><li> Iniciar o ciclo de novo, proporcionando tanto as garrafas de água e etanol em ratos, tal como descrito nas etapas 2.1 a 2.14. <ol><li> Repita esse ciclo por múltiplos (cerca de 12) sessões para induzir níveis elevados e estáveis ​​de consumo de etanol. Ver <strong>Figura 1</strong> para resultados representativos do consumo de etanol (g / kg) na gaiola de alojamento. </li><li> Alternar a posição espacial da garrafa de etanol e de água na tampa através de sessões de gaiola para evitar o desenvolvimento de um lado de preferência. </li><li> Faça uma solução de etanol fresco uma vez por semana para garantir a concentração de etanol preciso e estável em toda asessões. Minimizar o impacto da evaporação de etanol ao longo de períodos prolongados de tempo usando essa abordagem. </li></ol></li></ol><p class="jove_title"> 3 Aparelho </p><ol><li> Casa cada câmara condicionado dentro de um ventilado, cubículo atenuantes de som. </li><li> Construir cada câmara de condicionamento de um piso de haste de aço inoxidável metal, um teto de policarbonato transparente, policarbonato transparente parede traseira e porta da frente, e as paredes laterais do painel de metal de alumínio. <ol><li> Instalar uma porta de fluido centralmente perto da base da parede lateral direita de metal. Equipar a entrada para a porta de fluido com uma célula fotoeléctrica infravermelha para detectar a entrada na porta. </li><li> Conectar tubos de polietileno a partir da parte de trás da porta de fluido para uma seringa de 20 ml, contendo etanol, utilizando uma agulha de ponta romba. Colocar a seringa numa bomba de seringa, localizada fora do compartimento amortecedor de som. Para as sessões de treinamento discriminação de Pavlov, carregar etanol para a seringa antes de colocá-lo para dentro da bomba. Primeiro o tubo by avançar manualmente a bomba e limpe o excesso de etanol a partir do porto de líquidos. </li><li> Na lateral esquerda, instale um houselight branco central, perto do teto da câmara. </li><li> Instale um clicker (2 ciclo Hz) e um gerador de ruído branco com alto-falante no canto superior esquerdo da parede lateral esquerda. </li></ol></li><li> Equipe cada câmara condicionado com uma configuração específica de visuais, olfativas e táteis para criar um contexto distinto. <ol><li> Use dois contextos diferentes, designado Contexto 1 e Contexto 2 Configure metade das câmaras de condicionamento como Contexto 1 ea outra metade como Contexto 2. </li><li> Para criar um contexto, colocar painéis pretos de papelão sobre o teto e paredes frontal e traseira da câmara, odor spray de limão (10% v / v, feita através da diluição de óleo de limão na água da torneira) em uma placa de Petri e colocá-lo no centro de uma bandeja de resíduos de metal sob o chão da câmara, e inserir um painel claro piso de policarbonato sobre o metal rpiso od. Forre a bandeja de resíduos com papel absorvente ou forro de banco. </li><li> Para criar Contexto 2, deixar o tecto e as paredes da frente e de trás da câmara de descoberto, de modo que eles são claros, spray odor de amêndoa (10% v / v, preparada por diluição de benzaldeído em água da torneira) em uma placa de Petri, e colocá-lo centralmente em uma bandeja de resíduos de metais sob o chão da câmara, e inserir um painel perfurado chão de metal no chão haste de metal. Forre a bandeja de resíduos com papel absorvente ou forro de banco. </li></ol></li></ol><p class="jove_title"> 4. Habituação </p><p class="jove_content"> Habituar ratos para a sala de treino comportamental e cada um dos dois contextos dentro de câmaras de condicionamento. Para esta fase do experimento, não carregue o etanol em 20 ml seringas. </p><ol><li> Habituar ratos para o processo de ser transportados a partir da instalação de cuidados animais para a sala de treino comportamental no laboratório em duas sessões de habituação curtos. <ol><li> Na primeira sessão, carregar os ratos in suas gaiolas em uma carroça com rodas na unidade de cuidados de animais e transportá-los para a sala de treinamento comportamental em laboratório. Iluminar as luzes da sala, ligar os ventiladores em cada som atenuando cubículo, saia da sala, feche a porta e deixe os ratos ajustar a este novo ambiente por 20 minutos, mantendo-se em suas gaiolas no carrinho. No final deste período de 20 min, os ratos retornar para a instalação de cuidados animais. </li><li> Na segunda sessão, trazer os ratos para a sala de treino comportamental no laboratório. Como antes, deixe as luzes e os ventiladores cubículo na sala. Permanecer no quarto, com a porta fechada para minimizar as distrações, e manusear e pesar cada rato na sala de treinamento comportamental. Registre os pesos, e depois devolver os ratos para a instalação de cuidados com os animais. </li></ol></li><li> Habituar ratos para as câmaras condicionado em duas sessões de habituação curtas. <ol><li> Transportar os ratos para a sala de treinamento comportamental, gravar o peso de cada rato,e carregar cada rato na sua secção designada condicionado. </li><li> Na primeira sessão de habituação configurar metade das câmaras como Contexto 1 eo restante como Contexto 2. </li><li> Na segunda sessão de habituação configurar cada câmara com o contexto em que não foi utilizado na sessão anterior. </li><li> Realizar sessões de habituação, de forma contrabalançada, de tal modo que a metade dos ratos são primeiro expostos a um contexto, seguido de Contexto 2, e a outra metade são primeiro expostos a Contexto 2, seguido de Contexto 1. Idealmente, conduzir ambas as sessões de habituação na mesmo dia, embora possam ser repartidos ao longo de dois dias, se necessário. </li><li> Para as sessões de habituação, use um programa de computador para iluminar o houselight 1 min após o programa ter sido emitido. Registre o número total de entradas de porta fluidos feitas durante uma sessão de 20 min. No final da sessão, programar o houselight para desligar. Não apresenta nenhum etanol ou sugestões durante as sessões de habituação. </li></ol></li&gt; <li> Remover ratos das câmaras, devolvê-los às suas gaiolas, e transportá-los de volta para a unidade de cuidados animais. </li><li> Limpe o equipamento <em>(por exemplo,</em> painéis de chão, bandejas de lixo, placas de Petri). Veja o passo 5.7 para mais detalhes. </li></ol><p class="jove_title"> 5. pavloviano Discriminação Training (PDT) </p><p class="jove_content"> Ratos de trem para comportamentalmente discriminar entre um sinal sonoro que é emparelhado com etanol e um segundo sinal sonoro que não é. Para esta fase da experiência, encher as seringas de 20 ml com 15% de etanol. </p><ol><li> Equipe cada câmara condicionado com uma configuração específica de visual, olfativo e estímulos táteis para criar um contexto distinto, conforme descrito nas etapas 3.3.2 e 3.3.3 <ol><li> Atribuir metade dos ratos a um contexto e a outra metade para Contexto 2 de uma forma contrabalançado, com base no consumo de etanol e de preferência na média entre os dois últimos sessões do procedimento de acesso etanol gaiola de origem. Consulte a context usado durante PDT como Contexto A. NOTA: Cada rato só deve ter um contexto nesta fase de treinamento. </li><li> Certifique-se de que o estímulo olfativo é o último estímulo contextual para ser adicionado a cada câmara de condicionamento. Fechar as portas da câmara e o compartimento com atenuação de som que contém o estímulo olfativo e minimizar o comingling de odores diferentes. </li></ol></li><li> Traga os ratos na sala de treinamento comportamental, gravar o peso de cada rato, e carregar cada rato em sua câmara designado condicionado. </li><li> Inicie o programa de computador que controla a seguinte seqüência de eventos durante cada sessão PDT. <ol><li> No foi concedida em 5 min, após o programa, iluminar a houselight. </li><li> Apresentar o estímulo ruído branco eo clicker estímulo 16 vezes cada. Designar um destes estímulos auditivos a ser o CS + e o outro ser a CS, de um modo contrabalançada entre ambos os contextos. </li><li> Faça de cada CS + e CS julgamento last 10 seg. </li><li> Entregar 0,2 ml de etanol na porta de fluido durante a última 6 seg do CS +. Não entregar qualquer etanol durante o CS. </li><li> Faça com que cada sessão PDT últimos 58 min em média. Apresentar o CS + eo CS cada um em uma variável de tempo 67 seg programação. </li><li> Entradas de registros de fluidos porto durante o intervalo de 10 seg CS (SEC 10, durante apresentação CS), a 10 seg intervalo pré-CS (a 10 seg imediatamente antes da CS início), e os 10 s de intervalo pós-CS (a 10 seg imediatamente seguinte compensar CS), tanto para CS + e apresentações CS, bem como o total de entradas de porta fluido por toda a sessão. Registre também a duração de cada entrada de porta feito durante esses intervalos. </li><li> No final da sessão, desligue o houselight off. </li></ol></li><li> Remover ratos das câmaras, devolvê-los às suas gaiolas, e transportá-los de volta para a unidade de cuidados animais. </li><li> Verifique as portas de fluidos para assegurar que os ratos consumiram 3,2 ml de etanol entregues em cada PDT sessão. </li><li> Lavar o etanol para fora do tubo de polietileno com uma seringa de 60 ml contendo uma pequena quantidade de água. Em seguida, passar 60 ml de ar através do tubo usando um segundo, seringa vazia, para se certificar de que está seco. </li><li> Limpe o equipamento. Para a limpeza diária, lavar os painéis de chão, bandejas de resíduos e placas. Uma vez por semana, limpar toda a câmara de condicionamento. Não use produtos de limpeza perfumados para evitar a introdução de odores indesejados dentro do aparelho experimental. </li><li> Continuar sessões PDT diariamente até ratos mostram discriminação estável em suas respostas entrada de porta para o CS + vs CS. NOTA: As entradas de porta feitos em resposta ao CS + deve aumentar ao longo sessões, enquanto que as entradas de portas feitas em resposta ao CS deve permanecer baixa. O número exato de sessões PDT necessário para atingir discriminados condicionado responder pode variar, mas, como um guia, pensando em aproximadamente 20 sessões. </li></ol><p class="jove_title"> 6 Extinção </p><ol><li> Equipe cada cham condicionadober com visuais, olfativas e táteis para criar um contexto que é distinta da que os ratos experimentaram durante as sessões de PDT. Por exemplo, se um contexto foi utilizado em TFD, configurar a câmara de Contexto 2 para as sessões de extinção. Consulte o contexto utilizado durante a extinção em Contexto B. </li><li> Realizar sessões de extinção de forma idêntica para sessões PDT, mas não entregar qualquer etanol durante o CS +. <ol><li> Use o mesmo programa de computador que foi usado durante PDT para apresentar o + CS e CS. </li><li> Para manter a consistência entre as fases do experimento, carregar uma seringa vazia na bomba de seringa, e deixar a bomba ligada. Verifique se o tubo de polietileno não contém etanol antes das sessões de extinção. </li></ol></li><li> Prosseguir com as sessões de extinção diariamente até ratos já não mostram discriminados responder entre o CS + e CS. NOTA: As entradas de porta feitos em resposta ao CS + deve diminuir gradualmente ao longo das sessões de extinção, até que não haja diference entre entradas de porta feitos em resposta ao CS + e entradas de portos, realizado em resposta ao CS. O número exato de sessões de extinção necessário para extinguir condicionado responder pode variar, mas como um guia, pensando em aproximadamente 8 sessões. </li><li> Limpar o equipamento na extremidade de cada sessão, como descrito na etapa 5.7. </li></ol><p class="jove_title"> 7-Contexto induzida Renovação Teste </p><ol><li> Equipar cada câmara de condicionamento com os visuais, olfativas e táteis para criar o contexto que foi experimentado durante as sessões de PDT. Por exemplo, se uma dada ratazana experimentado Contexto 1 em sessões de PDT, configurar a câmara como um Contexto da sessão de teste. Consulte o contexto utilizado durante o ensaio renovação como Contexto A. </li><li> Realizar o teste de renovação induzida pelo contexto de forma idêntica para sessões de PDT, mas não entregar qualquer etanol durante o teste de renovação. Apresentar o <br /> CS + e CS como antes durante as sessões de PDT, mas reter o etanol. <ol><li> Use o mesmo pr computadorogram que foi usado durante PDT para apresentar o CS + e CS. </li><li> Para manter a consistência entre as fases, carregar uma seringa vazia na bomba de seringa, e deixar a bomba ligada. Certifique-se o tubo de polietileno não contém etanol, antes da sessão de teste. </li></ol></li><li> Observe renovação induzida pelo contexto do comportamento álcool-seeking pavloviano condicionado. NOTA: Este efeito de renovação será evidente pelo aumento seletivo do número de entradas de portos, realizado em resposta ao CS +, sem qualquer alteração no comportamento entrada de porta feito em resposta ao CS, em relação ao CS + e CS respondendo observado no final do treinamento extinção. </li><li> Limpar o equipamento no final da sessão, como descrito na etapa 5.7. </li></ol>

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<p class="jove_content"> Todos os procedimentos experimentais foram aprovados pelo Comitê de Ética em Experimentação Animal da Universidade Concordia e estão em conformidade com as diretrizes do Canadian Council on Animal Care. <span style="font-size: 14px; line-height: 28px;">A publicação de acesso livre deste artigo foi possível graças ao patrocínio da Med Associates Inc.</span> </p>

<p class="jove_content"> Os resultados deste processo revelam que os estímulos ambientais distintos que em geral acompanham a entrega de álcool pode adquirir a capacidade de dirigir o comportamento de busca do álcool. Eles também demonstram que estímulos contextuais associados com a disponibilidade ou ausência de álcool antes pode guiar condicionado respostas comportamentais a estímulos álcool preditivo discretos. </p><p class="jove_step"> As etapas críticas no âmbito do protocolo </p><p class="jove_content"> Contextos na presente tarefa incorporar estímulos que são externos ao rato. No entanto, o estado interoceptivo do rato em teste também pode constituir um &quot;contexto&quot; que poderia afetar <sup>de</sup> renovação <sup>de 20.</sup> Por isso, é importante habituar ratos para quaisquer procedimentos que possam ocorrer antes do teste renovação e mudar seu estado interoceptivo, como microinfusions intracranianas ou injeções sistêmicas. Além disso, a habituação ao microinfusions ou injeções sistêmicas devem ser realizadas antes de ambos extinção e PDT sessions, para impedir que tais procedimentos de se tornar associado com uma fase específica do experimento. </p><p class="jove_content"> Parâmetros temporais, como a duração do CS, intervalo CS-US, eo intervalo entre tentativas (ITI), têm sido conhecidos por influenciar condicionado responder em uma variedade de condicionamento paradigmas <sup>21-26.</sup> Da mesma forma, os parâmetros temporais podem influenciar a observação do efeito de renovação no nosso processo. Qualquer conjunto de outros que não os especificados no protocolo de parâmetros devem ser validadas experimentalmente em estudos-piloto antes de prosseguir para empregá-los em pesquisas mais amplas. </p><p class="jove_step"> Modificações e solução de problemas </p><p class="jove_content"> Pesquisa anterior tenha verificado que a quantidade de etanol que os ratos consomem na gaiola pode variar em função do fornecedor de que os ratos são comprados <sup>17,27.</sup> Fornecedor é, portanto, uma importante consideração para esta fase do estudo. De igual modo, a escolha da estirpe de ratos deve ser considered com cuidado, pois há inúmeros relatos de diferenças de tensão no consumo de etanol <sup>28-31.</sup> </p><p class="jove_content"> Trabalhos anteriores em um paradigma aversivo condicionado tem mostrado que a idade de desenvolvimento de ratos influencia a observação de renovação de um medo condicionado Pavloviana resposta <sup>32.</sup> A falta de um efeito de renovação em ratos muito jovens (menores de dia pós-natal 20) foi sugerido para ser prejudicada devido à codificação de informações contextuais <sup>33.</sup> A idade de desenvolvimento de ratos a serem utilizados no presente processo, deve ser tomado em consideração. </p><p class="jove_content"> Renovação de comportamento pavloviano álcool-seeking condicionado foi observado que 10% <sup>9,16,</sup> 15% de <sup>14,</sup> ou 20% <sup>15</sup> soluções de etanol foram utilizados durante o acesso etanol gaiola e fases PDT do experimento. Investigadores podem usar qualquer uma dessas concentrações de etanol para fins de estabelecer este procedimento em seu laboratories. </p><p class="jove_content"> Durante a aquisição de formação discriminação Pavloviana é importante verificar entradas de fluido depois de cada sessão para garantir que todo o etanol que foi entregue foi consumido. Deixando os ratos em câmaras de condicionamento para um adicional de 10 - 15 minutos depois de cada sessão pode ser feito cedo no PDT para encorajar o consumo de todo o etanol que não foi consumido durante a sessão. O número de ratos que saem etanol não consumido nas portas deve diminuir à medida que progride PDT. Se, no final de ratos PDT são (a) não consumir todo o etanol e / ou (b) não responder ao CS +, em seguida, estes assuntos devem ser descartados do estudo. A maioria dos ratos responder de forma confiável mais para a + CS do que o CS. No entanto, alguns ratos podem responder em níveis elevados para ambas as pistas, assim, não mostrando evidências de discriminação. Os ratos que mostram elevada respondendo tanto ao CS + e CS durante PDT não são descartados do estudo. Em nossa experiência, esses assuntos mostram discriminação notáveldurante a primeira sessão de extinção na ausência de etanol, o que sugere que os altos níveis de resposta para o CS durante a TFD são conduzidos pela presença de etanol na porta de fluido. Estes temas também tendem a mostrar efeitos de renovação robustos. </p><p class="jove_step"> Limitações do procedimento </p><p class="jove_content"> Porque a renovação de teste é conduzido na ausência de etanol, entradas de porta condicionado induzidos pelo CS + diminuir gradualmente em toda a sessão de teste. Portanto, o montante global de comportamento gerado pelo teste, contra a qual as mudanças induzidas podem ser avaliados, é bastante baixa. Porque nessa tarefa de responder tende a ser maior no início em relação ao final da sessão de teste, é importante examinar os efeitos de manipulações experimentais sobre condicionado respondendo a título de experiência-a-julgamento. </p><p class="jove_content"> As inscrições no receptáculo fluido fornecer a principal medida dependente nessa tarefa. No entanto, uma entrada de porta errada pode ser registered se um animal está suficientemente perto para o recipiente de fluido para quebrar o feixe de infravermelhos na frente da abertura da porta de fluido com os bigodes. Embora raras, as respostas erradas são óbvias como um aumento dramático na porta de entradas totais em relação ao comportamento do sujeito em sessões anteriores. Filmando os animais que facilitaria a detecção de possíveis respostas erradas, e permitir uma análise mais refinada dos comportamentos exibidos pelos ratos durante todo o treinamento e testes. </p><p class="jove_step"> Significância da técnica no que diz respeito a métodos alternativos existentes / </p><p class="jove_content"> Renovação induzida pelo contexto do comportamento droga-seeking é amplamente estudado utilizando uma tarefa condicionamento instrumental em que a variável dependente primária é uma resposta operante, tal como uma alavanca de imprensa <sup>8,34</sup> ou nariz picar <sup>12.</sup> No presente procedimento, a resposta de procura da droga é &quot;abordagem condicionado&quot; para a porta de fluido que é provocada por apresentaçõesa + CS. Esta resposta é provavelmente adquiridas através da aprendizagem pavloviana, que é o processo de aprendizagem que medeia a formação de associações entre estímulos ambientais e os efeitos farmacológicos das drogas de abuso em seres humanos. </p><p class="jove_step"> As aplicações futuras </p><p class="jove_content"> Esta tarefa pode ser utilizado em combinação com neuropharmacology, Optogenetics e neuroquímica para estudar os mecanismos neuronais que estão envolvidos na renovação induzida pelo contexto de comportamento Pavloviana-condicionado à procura de álcool <sup>16.</sup> Além disso, os mecanismos comportamentais e neurais que regulam a aquisição e extinção de comportamento álcool-seeking pavloviano-condicionado pode ser investigado. Por último, esta tarefa pode ser usado para explorar as manipulações de extinção que possam impedir a renovação, uma direção importante para a investigação translacional destinada a reduzir o impacto da droga associada contextos ambientais na reatividade a estímulos preditivos de drogas após a terapia de exposição aos estímulos em addi humanocts. </p>

<p class="jove_content"> Permanecer sóbrio é um desafio considerável enfrentado por pessoas que sofrem de transtornos de abuso de álcool. A abstinência é um período de vulnerabilidade ao impacto comportamental, psicológica e fisiológica de estímulos ambientais que habitualmente acompanham o uso de álcool, o que pode, através de condicionamento pavloviano, tornar-se associados com a embriaguez <sup>1,2.</sup> A exposição a sinais preditivos de álcool pode provocar respostas condicionadas, como desejo de álcool, que podem promover comportamentos de busca de álcool que facilitam recaída <sup>3,4.</sup> </p><p class="jove_content"> As seqüências estereotipadas de comportamento que levam ao consumo de álcool pode causar certos tipos de estímulos a serem rotineiramente experimentou imediatamente antes de os efeitos farmacológicos do consumo de álcool. Por exemplo, a visão, cheiro e sabor de álcool são propriedades sensoriais do álcool que antecedem confiavelmente intoxicação. Além desses sinais, que são referidos como &quot;discreto&quot; ou &quot;proximapistas de l &#39;, contextos ambientais em que as drogas são regularmente usados ​​também podem provocar ânsia <sup>5,6.</sup> A exposição a locais físicos em que as drogas foram anteriormente utilizados podem, portanto, ser um gatilho importante para a recaída <sup>7.</sup> </p><p class="jove_content"> Os modelos animais têm sido desenvolvidas para estudar os mecanismos neurais que mediam os efeitos de drogas sobre o comportamento associados contextos de procura de droga <sup>8-13.</sup> O procedimento aqui descrito permite a investigação de como os contextos associados ao consumo de álcool pode modular o comportamento de busca do álcool, que é provocada por um discreto, álcool preditivo sugestão pavloviano. </p><p class="jove_content"> Treino de discriminação pavloviano é conduzida em um contexto ambiental específico, onde os ratos são treinados para comportamentalmente distinguir entre dois estímulos auditivos condicionado, a + CS que está emparelhado com álcool e um CS que não é. Sessões de extinção são, então, realizado em um contexto diferente, onde respondendo ao CS+ Diminui como resultado do álcool a ser retido. Posteriormente, a reexposição ao álcool associado contexto de formação pavloviano desencadeia um aumento seletivo em comportamento de busca do álcool induzida pela CS +, sem alteração na resposta ao CS. Este resultado, que temos consistentemente replicados <sup>9,14-16,</sup> estende-se resultados de procedimentos instrumentais condicionado em que foram encontrados os contextos de drogas para estimular a renovação das respostas operantes associados com a entrega de drogas <sup>10,13.</sup> </p>

<table><tbody><tr><td>Heavy-duty utility cart</td><td>Rubbermaid</td><td>#4520-88</td><td>Used to transport rats in their home cage from the colony room to the behavior room or other facilities</td></tr><tr><td>Rats, Long-Evans</td><td>Harlan</td><td></td><td>Rats weighed 220-240 g on arrival</td></tr><tr><td>Ohaus scale</td><td>Fisher</td><td>#S402421</td><td>For weighing rats; tare the bottom of a plastic container first, then put the rat in it to weigh it</td></tr><tr><td>Beta-chips, aspen</td><td>Harlan</td><td>#7090A</td><td>Bedding for home cage</td></tr><tr><td>Nylabone</td><td>Bio-Serv</td><td>#K3580</td><td>Enrichment provided in the home cage</td></tr><tr><td>Autoclavable rodent chow</td><td>Agribrands, Charles River</td><td>#5075</td><td>For feeding rats</td></tr><tr><td>Wire bar cage lid</td><td>Ancare</td><td>#R20SS2B</td><td>Custom made with two slots for&amp;nbsp; bottles</td></tr><tr><td>Cage bottom</td><td>Ancare</td><td>#R20PC</td><td>To house rats</td></tr><tr><td>Stoppers</td><td>Ancare</td><td>#8.5</td><td>Rubber stoppers to occlude opening in water bottles &amp;amp; graduated cylinders; goes with ball point sipper tubes&amp;nbsp;</td></tr><tr><td>Ball point tubes, 1.5&quot; length</td><td>Ancare</td><td>#TD-99</td><td>Inserted in stoppers, enables rats to drink from water bottles and graduated cylinders</td></tr><tr><td>Water bottles</td><td>Ancare</td><td>#FSPC8HT</td><td>473 ml (16 oz) capacity, needs to be occluded with stopper</td></tr><tr><td>Graduated cylinders, 100 ml</td><td>Fisher</td><td>#0300741</td><td>To contain ethanol during alcohol exposure in the home cage; spouted end needs to be cut off to enable occlusion with stoppers</td></tr><tr><td>Mettler scale</td><td>Mettler Toledo</td><td>#MS6001S</td><td>For weighing water bottles and ethanol cylinders</td></tr><tr><td>Polyethylene tubing, 1/32&quot; inner diameter,&amp;nbsp; 1/32&quot; wall thickness, 3/32&quot; outside diameter</td><td>Fisher</td><td>#14-169-1A</td><td>To connect 20 ml ethanol syringe to fluid port</td></tr><tr><td>Needle, 18 g, 1.5&quot; length</td><td>Fisher</td><td>#B305199</td><td>To connect polyethylene tubing to ethanol syringes; needs to be filed</td></tr><tr><td>Syringes, 20 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#1482316J</td><td>To contain ethanol on the fluid pumps; needs to be connected to the polyethylene tubing by a 18 G 1 1/2 needle</td></tr><tr><td>Syringes, 60 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#14-820-11</td><td>To flush polyethylene tubing after PDT sessions</td></tr><tr><td>Single speed syringe pump, 3.33 rpm</td><td>Med Associates Inc.</td><td>#ENV-018MD</td><td>To hold 20 ml ethanol syringes and deliver ethanol</td></tr><tr><td>Fluid port</td><td>Med Associates Inc.</td><td>#ENV-254-CB</td><td>To enable ethanol delivery during PDT sessions; connected to ethanol syringes with polyethylene tubing</td></tr><tr><td>Port entry infrared detector</td><td>Med Associates Inc.</td><td>#ENV-215M</td><td>Fixed on both sides of&amp;nbsp; fluid port; need 2 per fluid port</td></tr><tr><td>Houselight, 28 V, 100 mA</td><td>Med Associates Inc.</td><td>#ENV-135M</td><td>Provides general lighting in conditioning chamber</td></tr><tr><td>Clicker module</td><td>Med Associates Inc.</td><td>#ENV-225SM</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>White noise generator with speaker</td><td>Med Associates Inc.</td><td>#PHM-100</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>Bar floor</td><td>Med Associates Inc.</td><td>#ENV-009A-GF</td><td>To go with modular test chamber</td></tr><tr><td>Bench coat, absorbent</td><td>VWR</td><td>#89126-790</td><td>Absorbent paper to put in metal tray, needs to be cut to fit the metal tray</td></tr><tr><td>Polycarbonate sheet, 1/8&quot; thick, 12-5/8&quot; x 10&quot;</td><td>Johnston Plastics</td><td>#30102515</td><td>To make smooth transparent floor insert used for context 1. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Aluminium sheet perforated, 0.063&quot; thick, 48&quot; x 96&quot;, 0.250&quot; holes x 0.375&quot; centers</td><td>Anica Steel</td><td></td><td>To make perforated floor insert used for context 2. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Black cardboard, 0.053&quot; thick, 28&quot; x 44&quot;</td><td>Omer DeSerres</td><td>#P1909</td><td>To construct panels covering the ceiling, door and back wall of the conditioning chamber in context 1. Needs to be cut to size. Each chamber needs three panels measuring approximately 11.5&quot; x 13.25&quot;. The panels can be joined by the narrower side using duct tape.</td></tr><tr><td>All-purpose strength duct tape, 1.88&quot; x 45 yd.</td><td>Duck Tape</td><td></td><td>Adhesive material to connect the black cardboard panels used to cover the back wall, ceiling and door in context 1.</td></tr><tr><td>Modular test chamber</td><td>Med Associates Inc.</td><td>#ENV-009A</td><td>Conditioning chamber</td></tr><tr><td>Sound attenuating cubicles</td><td>Med Associates Inc.</td><td>#ENV-200R3AM</td><td>Comes with installed 28 V DC fan (#ENV-025F)</td></tr><tr><td>Petri dish</td><td>Fisher</td><td>#08748B</td><td>Bottom only, to contain the sprayed solution that provides the odor stimulus; needs to be centered in the metal tray in conditioning chambers</td></tr><tr><td>PCI interface package</td><td>Med Associates Inc.</td><td>#DIG-700P2-R2</td><td>Connects tabletop interface cabinet to computer; includes 1 interface card, 1 Decode card, 1 ribbon cable and 1 28 V DC power cable</td></tr><tr><td>Large tabletop cabinet and power supply</td><td>Med Associates Inc.</td><td>#SG-6510D</td><td>Used to contain the 12 cards included in the SmartCrtrl 8 Input, 16 Output package</td></tr><tr><td>SmartCtrl 8 Input, 16 Output package</td><td>Med Associates Inc.</td><td>#DIG-716P2</td><td>Used to connect and control the devices in the conditioning chamber. The package includes all the cables, the interface module and the connection panel.</td></tr><tr><td>Med PC IV software</td><td>Med Associates Inc.</td><td>#SOF-735</td><td>Software to run programs. Needs to be installed on the computer.</td></tr><tr><td>Monitor LG, 19&quot; LCD</td><td>Concordia computer store</td><td>#W1942TQ-BF</td><td>Computer monitor; to go with computer running Med PC IV</td></tr><tr><td>HP Z200 tower workstation</td><td>Concordia computer store</td><td>#BZ788US#ABA</td><td>Computer on which Med PC IV is run</td></tr><tr><td>Benzaldehyde, &amp;ge;99%</td><td>Les produits chimiques OMEGA Chemical Company Inc.</td><td>#B37-50</td><td>Dilute in tap water for almond context odor</td></tr><tr><td>Lemon oil, cold pressed, California</td><td>Sigma Aldrich</td><td>#W262528-1KG-K</td><td>Dilute in tap water for lemon context odor</td></tr><tr><td>Ethyl alcohol, 95%</td><td>Commercial Alcohols</td><td></td><td>Dilute in tap water to desired concentration</td></tr></tbody></table>

a procedure to observe context-induced renewal of pavlovian-conditioned alcohol-seeking behavior in rats

<p class="jove_content"> Contextos ambientais em que as drogas de abuso são consumidos podem desencadear o desejo, a resposta subjetiva pavloviano-condicionado que pode facilitar o comportamento droga-seeking e rápida recaída em usuários de drogas em abstinência. Temos desenvolvido um procedimento para estudar os processos comportamentais e neurais que medeiam o impacto do contexto sobre o comportamento de procura de álcool em ratos. Após a aclimatação ao paladar e efeitos farmacológicos de etanol a 15% na gaiola, ratazanas Long-Evans machos receber formação discriminação Pavloviana (PDT), em câmaras de condicionamento. Em cada sessão diária (de segunda a sexta) PDT, 16 ensaios de cada um dos dois estímulos diferentes de 10 segundos auditivo condicionado ocorrer. Durante um estímulo, o CS +, 0,2 ml de etanol a 15% é fornecido a uma porta de fluido para consumo oral. O segundo estímulo, o CS, não está emparelhado com etanol. Entre as sessões, as inscrições para o porto de líquidos durante o aumento CS +, enquanto que as entradas durante o CS se estabilizar em um nível mais baixo, indicando que a predictive associação entre o CS + e o etanol é adquirido. Durante PDT cada câmara está equipada com uma configuração específica de estímulos contextuais visuais, olfativas e táteis. Seguindo PDT, formação de extinção é realizado na mesma câmara que já está equipado com uma configuração diferente de estímulos contextuais. O CS + e CS são apresentados como antes, mas o etanol é retido, o que causa um declínio gradual na entrada do porto durante o CS +. No momento do teste, os ratos são colocados de volta no contexto PDT e apresentados com o CS CS + e, como antes, mas sem etanol. Esta manipulação desencadeia um aumento robusto e seletiva do número de entradas de porta feitos durante o álcool preditivo CS +, sem alteração na resposta durante o CS. Este efeito, referido como renovação induzida pelo contexto, ilustra a capacidade potente de contextos associados com o consumo de álcool para estimular o comportamento de procura de álcool, em resposta a sinais de álcool de Pavlov. </p>

<p class="jove_content"> Acesso etanol intermitente na gaiola: O consumo de etanol (g / kg) e preferência aumento de etanol através de sessões de acesso etanol intermitente na gaiola, geralmente atingindo níveis estáveis ​​dentro de 8-12 sessões <strong>(Figura 1).</strong> No conjunto de dados mostrado na <strong>Figura 1,</strong> o consumo de etanol estável na média entre as duas últimas sessões de acesso etanol gaiola variou 1,33-6,44 g / kg para os ratos individuais. A fase de consumo de etanol gaiola do experimento pode ser interrompida assim que o g / kg estacionou o consumo, e os ratos são mantidos neste nível estável de beber por um certo número de sessões. </p><p class="jove_content"> Para analisar os dados desta fase, realizar separado análise de medidas repetidas de variância (ANOVA) através do fator intra-sujeito da sessão de consumo de etanol (g / kg) e de preferência etanol. </p><p class="jove_content"> Esquemática do procedimento de renovação induzida pelo contexto: Cada fase doprocedimento experimental (PDT, extinção, teste de renovação) é ilustrada na <strong>Figura 2.</strong> Veja a figura legenda para mais detalhes. </p><p class="jove_content"> Resposta medidas e análise de dados: Em todas as fases comportamentais descritas a seguir, as respostas entrada de porta feitos durante o CS + e CS são normalizados em relação às respostas entrada de porta de linha de base pré-CS. Para criar as medidas CS normalizados, subtraia o número de entradas de portas feitas durante o intervalo da linha de base pré-CS (a 10 seg imediatamente antes da CS início) a partir do número de entradas de porta feitas durante as apresentações do CS + e CS. A normalizada CS + e medidas cs- normalizados servem como indicadores de comportamento provocada especificamente por apresentações CS, elevado acima dos níveis basais. </p><p class="jove_content"> Realizar medidas repetidas separadas ANOVA com os fatores assunto dentro repetidas de sessão e tipo CS (CS + vs CS) para o PDT e fases de extinção. Para as comparações entre fase (PDT, extinção, renovarteste al), os dados de uso de média entre as duas últimas sessões de PDT e extinção ea uma sessão de dados de teste de renovação em medidas repetidas ANOVA. Para acompanhar efeitos significativos principais e interações, t-testes uso <em>pós hoc</em> para amostras independentes ou pareadas que são corrigidas para comparações múltiplas. Critério de significância foi fixado em α = 0,05. </p><p class="jove_content"> PDT: respostas entrada da porta ao aumento CS + em todas as sessões PDT, ao passo que as respostas entrada portuária à CS- permanecer baixa e estável, de modo geral. Note-se que as respostas entrada da porta para o CS pode aumentar ligeiramente em primeiras sessões de treinamento PDT; no entanto, entradas de porta feitos em resposta ao CS estabilizar a um nível muito mais baixo do que as feitas em resposta ao CS +. Discriminação de sucesso é evidenciado por um aumento estatisticamente significativo nas entradas da porta durante o CS + em comparação com o CS <strong>(Figura 3).</strong> Este resultado indica que o comportamento álcool-seeking pavloviano condicionado tem seren estabelecida para o CS +, mas não o CS. </p><p class="jove_content"> Extinção: respostas entrada da porta ao CS + diminuir gradualmente ao longo das sessões de extinção. Respostas entrada de porta para o CS permanecer baixa e estável em todas as sessões de extinção. Idealmente, discriminados responder é abolida até o final do treinamento extinção, como evidenciado pela falta de uma diferença estatisticamente significativa entre as entradas de portas para o CS + e CS. No entanto, não é incomum para um certo grau de discriminação de responder a permanecer, até ao final da formação de extinção. Neste caso, é importante verificar que as respostas de entrada da porta para o CS + no final do treino de extinção são estatisticamente menor do que as respostas de entrada de porta para o CS + no início de extinção, bem como no final da PDT <strong>(Figura 3)</strong> . Este padrão de resultados indica que o comportamento álcool-seeking pavloviano condicionado foi extinto de forma significativa. </p><p class="jove_content"> Teste renovação induzida por Contexto: Portrespostas de entrada para o aumento CS + no teste de renovação induzida pelo contexto, em relação a entradas de porta CS + observados no final do treinamento extinção. Este aumento é seletiva para o CS +, como entradas de porta para o CS permanecem inalterados. Além disso, as entradas de portas para o CS + são estatisticamente elevados em comparação com as entradas da porta para o CS no ensaio de renovação induzida pelo contexto <strong>(Figura 4).</strong> Confirmação estatística da comparação das entradas de porta CS + entre extinção e teste, bem como comparações de CS + e entradas de porta CS dentro de teste, indica uma renovação seletiva de comportamento pavloviano condicionado em busca de álcool induzida pelo contexto álcool associado ao teste. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 1" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig1highres.jpg" width="400" /><br /> <strong>Figura 1 Escalonamento de consumo de etanol e de preferência induzida por etanol acesso intermitente na gaiola.</strong> Média &amp;# 177; SEM <strong>(A)</strong> consumo de etanol (g / kg) e <strong>(B),</strong> de preferência o etanol através de 12 sessões de acesso etanol intermitente na gaiola. Ambas as medidas tendem a aumentar em todas as sessões e depois estabilizar. Consumo de etanol estável na média entre as duas últimas sessões de acesso etanol gaiola variou 1,33-6,44 g / kg para ratos individuais. Estes dados foram obtidos em ratos Long-Evans machos potável de 15% (v / v) de etanol. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 2" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig2highres.jpg" width="400" /><br /> <strong>. Figura 2 Um esquema do procedimento experimental</strong> O procedimento consiste em três fases: formação discriminação pavloviano (PDT), extinção e teste de renovação induzida pelo contexto. Durante cada sessão PDT, os ratos recebem 16 ensaios de um estímulo auditivo condicionado (CS +) que assinala a entrega de etanol e 16 ensaios deum segundo estímulo auditivo (CS) que não está associado com a entrega do etanol. Sessões de PDT, ocorrem num contexto A, composto por uma configuração específica de visual, olfativo e estímulos táteis. Durante as sessões de extinção, que são realizados em Contexto B, composto por um conjunto diferente de estímulos visuais, olfativos, táteis e, o CS + e CS são apresentados, mas o etanol é retido. Para o teste de renovação induzida pelo contexto, os ratos são devolvidos ao Contexto A, eo CS + e CS são apresentadas sem etanol. <a href="https://www.jove.com/files/ftp_upload/51898/51898fig2highres.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 3" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig3highres.jpg" width="400" /><br /> <strong>Figura 3 Aquisição e extinção da discriminados pavlovianas condicionado responder para o etanol.</strong> Média ± SEM entradas de porta normalizada made durante o CS + e CS através PDT (sessões 1-18) no contexto de um e de extinção (sessões 19-26) em Contexto B. Estes dados foram obtidos utilizando ratos Long-Evans do sexo masculino, treinado com 10% (v / v) etanol como o estímulo incondicionado (US), que foi apresentado em associação com a + CS durante PDT. * P &lt;0,05, CS + contra CS. Figura publicado originalmente em Chaudhri, Sahuque, e Janak (2008). Usado com permissão da Elsevier. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 4" fo:content-width="5in" src="/files/ftp_upload/51898/51898fig4highres.jpg" width="500" /><br /> <strong>Figura 4 renovação de</strong> entradas <strong>pavlovianas condicionado responder para o etanol.</strong> Média ± SEM porta normalizada feitas durante o CS + e CS em sessões PDT no contexto de um, sessões de extinção no Contexto B, e teste de renovação em Contexto A. Os dados são <strong>induzidas por Contexto</strong> rendimento médio das duas últimas sessões de PDT e extinção; o teste de renovação é uma única sessão. Estesos dados foram obtidos em ratos machos Long-Evans, formados com 10% (v / v) de etanol como o estímulo não condicionado (US), que foi apresentado em associação com o CS + durante a PDT. * P &lt;0,05; ** P &lt;0,001, CS + contra CS. #p &lt;0,05, CS + PDT contra CS + extinção. ^ P &lt;0,05, CS + extinção contra teste CS +. Figura publicado originalmente em Chaudhri, Sahuque, e Janak (2008). Usado com permissão da Elsevier. </p>

Watch this Scientific Journal Video about A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats at JoVE.com

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

<p class="jove_content"> Um procedimento para o estudo da capacidade de um álcool associado contexto ambiental para desencadear a renovação do comportamento de procura de álcool em ratos, está descrito. </p>

Um procedimento para Observar Renovação induzida por Contexto de pavloviano condicionado Comportamento Álcool-seeking em Ratos

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

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12.8K visualizações.  Concordia University.  Um procedimento para o estudo da capacidade de um álcool associado contexto ambiental para desencadear a renovação do comportamento de procura de álcool em ratos, está descrito. 

Vídeo: A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Esterification

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">First, put on a lab coat, safety glasses, and nitrile gloves.<strong> Note:</strong> Some of the reagents are volatile or have unpleasant odors, so you will handle them in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Ensure that you have glass and organic waste containers and that each sink has paper towels.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Follow the table to designate codes for the carboxylic acids and alcohols used in this lab. The carboxylic acids are coded A through C, and the alcohols are coded D through I. The students will use a 3:1 molar ratio of alcohol to carboxylic acid in the reaction.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Code</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Compound</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>MW<br />
					(g/mol) </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Density at RT (g/cm<sup>3</sup>)</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Moles per vial</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Grams per vial</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>cm<sup>3</sup> per vial </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Number of vials</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">A</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">60.05</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1.049</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">4.2035</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">4.0</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">B</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">88.11</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.953</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.1677</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.5</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">C</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Anthranilic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">137.14</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">-</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">9.5998</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">-</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">D</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Propanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">60.10</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.803</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">12.6210</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">15.7</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">E</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl alcohol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">88.15</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.810</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">18.512</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">22.8</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">F</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Butanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">74.12</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.810</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">15.565</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">19.2</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">G</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">46.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.789</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">9.6747</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">12.3</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">H</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">32.04</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.791</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.7284</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">8.5</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">2</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">I</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Octanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">130.23</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.830</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">27.348</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">32.9</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare the three carboxylic acids: acetic acid, butyric acid, and anthranilic acid. Each group will need 0.07 moles of their assigned carboxylic acid in a vial.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Tare a 10-mL glass vial on an analytical balance in a fume hood. Use a Pasteur pipette to measure 4.2035 g of acetic acid, which will be about 4 mL. Cap the vial and label it &#8216;A&#8217;. Prepare two more labeled vials of acetic acid in the same way.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Weigh 6.1677 g of butyric acid, which will be about 6.5 mL, in a tared 10-mL vial. Cap the vial and label it &#8216;B&#8217;. Prepare two more vials of butyric acid in the same way.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a small spatula to weigh 9.5998 g of solid anthranilic acid in a tared 10-mL vial. Cap the vial and label it &#8216;C&#8217;. You should now have a total of seven vials of carboxylic acid.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare the 7 alcohols. Each group will need 0.21 moles of their assigned alcohol in a vial. Use the table as a guide to prepare and label one vial of each alcohol. Prepare and label two vials of methanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">You should now have a total of seven vials of alcohol. Keep the reagent vials in the instructor&#39;s hood for now and put away the stock bottles of the carboxylic acids and alcohols.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Assign each student group one of these seven combinations of reagents.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Ester code</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Carboxylic acid </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Alcohol</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Ester product</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Scent</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AD</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Propanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Propyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Pear</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AE</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl alcohol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Banana</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AI</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Octanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Octyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Orange</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BF</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Butanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Pineapple</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BG</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Strawberry</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BH</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Apple</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">CH</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Anthranilic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methyl anthranilate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Grape</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Prepare or obtain seven vials of the fruit scents and label them with the fruit names as references.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Put bottles of saturated sodium bicarbonate and 3 M sulfuric acid in a dedicated fume hood, along with a roll of plastic paraffin film and scissors.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Set out filter paper and Pasteur pipettes in a central area so that every student group can easily access them.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;3-prong clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab jack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Heating mantle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Temperature controller</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pieces of tubing (for condenser)</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Joint clip</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Tube of vacuum grease</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Box of laboratory wipes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;10-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL round-bottom flask and flask stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Standard condenser</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pasteur pipette bulbs</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Just before the lab, fill a large insulated cooler with crushed ice. Place the cooler and an ice scoop in a central area in the lab.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">When the students arrive, show them how to safely waft vapors before giving them their assigned reagents.</div>
		</li>
	</ul>
	</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Prep

Esterificação

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

	Preparation of the LaboratoryExpand
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Education

JoVE Lab Manual

Química

Lab Manual Sub Articles

Identifying Alcohols

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Ferric Chloride Test for Phenols<button class="expand">Expand</button>
	<p>In this lab, you will identify an unknown alcohol using the ferric chloride test, the Jones test, and the Lucas test. You&#39;ll test known alcohols alongside the unknown alcohol as examples of positive and negative results for each test.</p>
	<p>The four known alcohols are 1-butanol, a primary alcohol, 2-butanol, a secondary alcohol, 2-methyl-2-propanol, a tertiary alcohol, and phenol. The four possible unknown alcohols are 1-propanol, 2-propanol, 2-methyl-2-butanol, and para-chlorophenol.</p>
	<p>You&#39;ll start with the ferric chloride test for the presence of phenols. Iron(III) chloride, or ferric chloride, forms a brown complex when it&#39;s dissolved in water. If you add a phenol to this solution, a purple iron(III)-phenol complex will form instead. This effect is not observed with non-aromatic or aliphatic alcohols.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-4">
		<div class="lab-step">Put on a lab coat, safety glasses, and gloves before you begin. <strong>Note:</strong> The alcohols that you will test are toxic and flammable, and the reagents that you will use are toxic and corrosive, so you will perform this lab in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Now, get your assigned unknown alcohol from your instructor and bring it to your fume hood. Label a 250-mL beaker &#8216;aqueous metal waste&#8217;.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, put five small test tubes in a rack and label them &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, &#8216;phenol&#8217;, and &#8216;unknown&#8217;. Bring the rack to the analytical balance to obtain phenol.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">If your unknown is a solid, bring it with you. Weigh about 0.2 g of phenol and pour it into the corresponding tube. Do the same for your unknown if it is a solid and then return to your hood.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now label three small vials &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, and &#8216;2-methyl-2-propanol&#8217;.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Bring the vials to the solvent hood and use clean Pasteur pipettes to fill each vial with about 1 mL of the matching alcohol.<strong> Note: </strong>Do not reuse pipettes.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Next, obtain at least four Pasteur pipettes and a 250-mL beaker. Get more pipettes whenever you need them, and don&#39;t put used pipettes back in the beaker.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">In your fume hood, pipette 3 &#8211; 4 drops of 1-butanol, 2-butanol, and 2-methyl-2-propanol into the corresponding tubes.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">To the phenol tube, add several drops of deionized water and shake to mix. This will provide better visual results for the ferric test.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">If your unknown alcohol is a liquid, use a clean pipette to place 3 &#8211; 4 drops of it in the unknown test tube.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, obtain the bottle of iron(III) chloride solution and add 2 &#8211; 3 drops to each test tube.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Stir each mixture well with a glass rod. Use deionized water to rinse off residual alcohol before mixing the contents of the next tube. Make sure that the phenol dissolves completely along with the unknown if it is a solid.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Wait 5 min before comparing your unknown to the known solutions. <strong>Note:</strong> If your unknown solution is purple, like the phenol solution, then your unknown is a phenol derivative. If it looks like the other three tubes, it is not a phenol derivative. Record the results of the test in your lab notebook.
		<h4><strong>Table 1: Identifying Unknown Alcohol</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Alcohol</strong></td>
					<td style="text-align: center;"><strong>Ferric Chloride Test</strong></td>
					<td style="text-align: center;"><strong>Jones Test</strong></td>
					<td style="text-align: center;"><strong>Lucas Test</strong></td>
				</tr>
				<tr>
					<td>1-butanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2-butanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2-methyl-2-propanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Phenol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Unknown</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/CDNSource/lm/labs/71/Table_1._Identifying_unknown_alcohol.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">The next tests don&#39;t work with phenols, so you&#39;ll only test aliphatic alcohols for the rest of the lab. If your unknown is a phenol derivative, skip the instructions for the unknown and test only the three known aliphatic alcohols.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">The Jones Test for Aliphatic Primary and Secondary Alcohols<button class="expand">Expand</button>
	<p>In this section, you&#39;ll perform the Jones test for primary and secondary alcohols. Jones&#39; reagent is made with chromium trioxide and sulfuric acid in water, which forms chromic acid (H<sub>2</sub>CrO<sub>4</sub>)<em> in situ</em>. This powerful reagent oxidizes secondary alcohols to ketones, primary alcohols to aldehydes, which after forming an aldehyde hydrate, are further reduced to carboxylic acids.</p>
	<p>The oxidation state of chromium is the key to this test. Chromium is in the +6 oxidation state in the Jones&#39; reagent. The Cr(VI) complexes in the reagent give it its bright reddish, orange color.</p>
	<p>When Cr(VI) oxidizes an alcohol, chromium is reduced to the +3 oxidation state. First, the alcohol and chromic acid form a chromate ester. Then, a base (H<sub>2</sub>O) cleaves the C-H bond of the alcohol, forming the carbonyl group while reducing Cr(VI) to Cr(IV). Because the carbon of the alcohol undergoes a 2-electron oxidation, and Cr(VI) a 2-electron reduction, this step is a reduction-oxidation step. Cr(IV) participates in further oxidation steps and is eventually reduced to Cr(III). Cr(III) is often present as hexaaquachromium(III) ions&#160;&#8212; [Cr(H<sub>2</sub>O)<sub>6</sub>]<sup>3+</sup> &#8212; and Cr(III) complexes, where H<sub>2</sub>O molecules are replaced by one or more sulphate ions, [Cr(H<sub>2</sub>O)<sub>5</sub>(SO<sub>4</sub>)]<sup>+</sup>. These complexes give Cr(III) the characteristic green color. So, when you mix Jones&#39; reagent with a primary or secondary alcohol in acetone, the orange solution will turn green.</p>
	<p>Jones oxidation of alcohols doesn&#39;t work with tertiary alcohols because the -OH group is already bonded to three carbon atoms and cannot form an extra C-O bond. Thus, you won&#39;t see a color change if you combine Jones&#39; reagent with a tertiary alcohol because chromium isn&#39;t reduced.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label four clean test tubes &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, and &#8216;unknown&#8217; and place 10 drops of acetone in each. Acetone is the preferred solvent for Jones oxidation.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, use clean pipettes to add 3 &#8211; 4 drops of the known aliphatic alcohols and your unknown alcohol to the corresponding tubes.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Stir each solution well with the glass rod and rinse the rod with water after mixing each one.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Now, obtain the bottle of Jones&#39; reagent and carefully add 2 &#8211; 3 drops to each tube. The reagent is strongly acidic and corrosive, so be careful with it and tightly cap the bottle when you&#39;re done. Then, mix each solution well, cleaning the glass rod after each one.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Let the solutions react for 10 min, and then compare your unknown to the knowns. <strong>Note:</strong> If the unknown solution turned green, like 1-butanol and 2-butanol, the test is positive for a primary or secondary alcohol. If it stayed orange, like 2-methyl-2-propanol, the test is negative.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Record the results in your lab notebook.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">The Lucas Test for Classifying Small Saturated Alcohols<button class="expand">Expand</button>
	<p>The Jones test doesn&#39;t distinguish between primary and secondary alcohols, so you&#39;ll use the Lucas test to identify which is which.</p>
	<p>Lucas&#39; reagent, which is a mixture of zinc chloride and hydrochloric acid, converts secondary and tertiary alcohols to chloroalkanes at room temperature. Chloroalkanes are nearly insoluble in water, so a positive result appears as the mixture separates into a cloudy chloroalkane-containing layer over a clear layer.</p>
	<p>The rate-determining step of the reaction involves converting the alcohol to a carbocation, so the speed of the reaction depends on how stable the carbocation is.</p>
	<p>Tertiary carbocations are very stable, so tertiary alcohols give a positive reaction almost immediately. Secondary carbocations are less stable, so secondary alcohols give a positive result after a few seconds to a few minutes. Primary carbocations are too unstable for this reaction, so primary alcohols give a negative result.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-5">
		<div class="lab-step">Now, label for clean test tubes as &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Obtain the bottle of Lucas&#39; reagent and use a graduated cylinder to measure 2 mL of the reagent for each tube.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Next, add 2 drops of 2-methyl-2-propanol to the appropriate tube. Immediately, stir the mixture for a few seconds and place it in the rack. The mixture will promptly separate into a clear lower layer and a cloudy upper layer.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Thoroughly rinse the glass rod and record the immediate positive result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Mix the remaining alcohols with Lucas&#39; reagent in the same way, using a fresh pipette for each alcohol and rinsing the glass rod each time. <strong>Note:</strong> Don&#39;t add too much alcohol or jostle the tubes because that can slow down the reaction.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Watch carefully after mixing the unknown alcohol with Lucas&#39; reagent to ensure that you can distinguish an immediate positive result from a positive result after a brief delay.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Check on the tubes every few minutes and record positive results as you see them. Note approximately how long it took for delayed positive results to appear.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Once the tubes have sat for at least 15 min, you can assume that the results are final. Record the results for 1-butanol, 2-butanol, and for your unknown if applicable.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">Now, let&#39;s clean up. Your instructor will collect the Cr(VI) solutions, so leave any tubes of orange solution from the Jones test in your hood.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">Empty the other test tubes into the waste beaker and rinse them with deionized water. Pour the waste into the aqueous metal waste container using a funnel and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Now, wash your glassware as usual and dispose of used pipettes in the glass waste. Lastly, clean your hood and throw out any trash in the lab trash container.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Identify your unknown alcohol. The four possibilities are 1-propanol, 2-propanol, 2-methyl-2-butanol, and para-chlorophenol.</p>
	<ul class="lab-items">
		<li data-sub-note-item="4-2">
		<div class="lab-step">Consider the results of the ferric chloride test first. If the mixture with your unknown alcohol turned purple, like phenol did, your alcohol must be para-chlorophenol. If you saw a negative result, your unknown is one of the three aliphatic alcohols.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">Look at the results of the Jones test. If the mixture with the unknown alcohol turned green, the unknown could be primary or secondary. If the mixture with your unknown alcohol stayed orange, your unknown alcohol is tertiary. Therefore, a negative Jones result means that your alcohol must be 2-methyl-2-butanol.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Lastly, look at the results of the Lucas test. If you had a negative result in the Jones test, you should also have seen an immediate positive result in the Lucas test. This confirms that your alcohol was tertiary. If your unknown had a positive result after a short delay, then your unknown alcohol is secondary, and therefore, must be 2-propanol. Finally, if your unknown had a negative result in the Lucas test, your alcohol is primary, and therefore, must be 1-propanol.</div>
		</li>
	</ul>
	</li>
</ol>

Identifying Alcohols: Ferric Chloride Test, Jones Test, and Lucas Test - Procedure

Identificação de álcoois

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

	Ferric Chloride Test for PhenolsExpand
	In this lab, you ...

Beer's Law

Beer-Lambert Law and Equilibrium Constant Determination - Procedure

Lei da Cerveja

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

	Lab Setup for Beer&#8217;s LawExpand
	In this lab, you will combine aqueous solutions of FeCl3 ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</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">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</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>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</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 style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</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 style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</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 style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</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/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

Dependência de Concentração

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

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

Optimal Foraging

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

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

Forrageamento ideal

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

Biologia

Ecologia

Animal Behavior

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

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

Comportamento animal

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

Extinction

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

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

Extinção

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

Group Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">The Hawk-Dove Game
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe hypothetical costs and benefits of the antagonist or cooperative behavior respectively. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Before the activity starts, review the cost-benefit table so that you understand how to score the game. <a href="https://www.jove.com/CDNSource/lm/labs/33/Table_1_Group_behavior.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">HYPOTHESES: The experimental hypothesis for this exercise could be that the dove strategy will become more frequent between repeated partners, and the hawk strategy will become more frequent when interactions are with a variety of partners. The null hypothesis could be that there will be no change in strategy over time with repeat or different partner combinations. NOTE: Benefit can be determined by looking at the left column and choosing the row that corresponds to the card you played. The box that corresponds to the choice of your partner can be found in the top row. For the purposes of this game, B equals four and C equals three. So depending on the cards played, player one here, which is you, would gain a score of two, four, zero or one half. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Collect a set of two game cards. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Take out the cards labeled hawk and dove and place them face down on the table or conceal them in your hands. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the instructor gives the signal show either of the two cards to the student sitting across from you. Try to strategize how to receive the highest benefit from every interaction on your own but do not communicate with your partner. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the Single Partner Recording Table (Table 2) provided by your instructor, note the cards played as well as the benefit you received for that round.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat showing cards to the same partner and determining benefit on your instructor&#39;s signal for a total of 15 rounds.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">At the end of those rounds, the student with the highest total benefit value was determined to be the &#8220;winner&#8221;, or the most fit.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">As a class, determine the percentages of hawks and doves played as well as the average benefit received for each round, using this formula: Class average benefit=(sum total benefit)/(number of players)
</div></li><li data-sub-note-item="1-11"><div class="lab-step">For the second game, and then for each round of play, one row of students should rotate to their right, leaving all players with a new partner.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Continue playing for 15 rounds or until students end up back in their starting positions, recording the data in the Multiple Partner Recording Table (Table 3) given to you by your instructor.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">For each round, determine the percentages of hawks and doves played that round, as well as the average benefit received.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Cheating and Cooperation Exercise
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this activity, you will exchange benefits, represented by colored or similar items, with your classmates. In this simulation, a cheater is defined as a person who gives fewer benefits than they receive. During this activity, you will observe the frequency of cheating behavior over time. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that cheaters will receive fewer benefits over time and cooperators will continue to provide equal benefits to one another. The null hypothesis is that the frequency of cheaters and cooperators does not change over time. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Each person should collect beads of one color to represent a tangible benefit, as well as one paper bag and a three-ring binder.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Get into a group of three so that all members have objects of the same color.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now find another group with a different color to pair with. In each round, partners will exchange benefits without knowing what they are receiving from their partner and without discussion within their group.  NOTE: Benefits of a different color from your starting color are worth twice as much.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Turn face to face with a member from the other group while still being able to conceal the objects in your possession.  TIP:<strong> </strong>Setting up a screen using a notebook or sitting behind a desk can be helpful here.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When given a signal from the instructor, place zero to two benefits into the paper bag and pass it to your partner.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Simultaneously, receive the bag from your partner and then remove the benefits from the bag.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Make a note of how many benefits you gave away and how many you received in the Cheating and Cooperation Table (Table 4) provided by your instructor.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Turn to a new member from the other group and repeat the exchange.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Rotate through all of the members of the other group for as many rounds as the instructor determines - which will not be revealed until the game ends so as not to alter player behavior.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Once the game has ended, note for each round in the cheating and cooperation table whether you gave more than your partner, received more from them or if you gave the same amount.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Now total up all of the benefits in your possession, with benefits you&#39;ve received from the other group being worth twice as much.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">For each round, the whole class should total up the number of cheaters - with cheaters defined as those who gave less than they received.
</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">For both the single partner hawk-dove game and the multiple partners hawk-dove game, use the whole class data to plot the percentages as two separate lines - one for the percentage of hawks, and one for the percentage of doves, for each of the 15 rounds.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Take note of whether the cards played changed as more rounds were played.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Manipulate the values for B and C to see if you can find the point at which values of B and C would render the hawk strategy no longer effective (this would be the point when there is no benefit to choosing the hawk strategy over always choosing the dove strategy).
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As a class, use the data from the cheating and cooperation activity to make a graph showing the percentage of rounds each individual player cheated versus the amount of benefits they ended with.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Consider whether it is better to be a cheater or a cooperator in this scenario and discuss as a class what makes this scenario different from the hawk-dove game.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Now, use the class data to graph the percentage of cheaters against the number of rounds, and look for any trends over time.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Consider which type of strategy would most likely evolve in a setting where partners interact repeatedly.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Then, note which tactic might be beneficial for a species that relies on cooperation. 
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Discuss as a class what would likely happen if you increased or decreased the relative value of the benefits you receive.
</div></li></ul></li></ol>

Group Behavior: Hawk-Dove Game and Trading Exercise - Procedure

Comportamento do grupo

The Hawk-Dove Game
ExpandNOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe ...

Artificial Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing Artificial Selection in Plants 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment, collect five two-week old grown plants from the generation one population that appear healthy. Note: Plants should appear green, not wilted, and not have damaged leaves. Experimental Hypothesis: The experimental hypothesis for this lab might be that the number of lead trichomes varies naturally, but the average number will increase over generations if only plants with the highest number of trichomes are interbred or decrease if those with the least are interbred. Null Hypothesis: The null hypothesis might be that the average number of trichomes will not change over subsequent generations after artificial selection.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Label each plant with a letter from A to E and with both partners&#8217; names.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Record any natural differences you see between your plant samples. Note: All of the plants are the same age, so any observed variation is due to expressed phenotype, not development.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use a hand lens to count the number of trichomes found on the petiole of the lowest leaf, not including the round, first-grown cotyledons for each plant. Note: It can be helpful to shine a light onto the plant and to hold it against a dark background, such as a lab bench, during the trichome analysis. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Both partners will conduct counts on each plant independently and compare answers. If the counts are different, enlist a third counter or take the average of the two results.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After this, determine the median number of trichomes for the entire class.  <a target="__blank" style="display:block;padding:20px;" href="https://www.jove.com/CDNSource/lm/labs/2/Lab_2_Table_A.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Determine the top and bottom 10% of the plants from the class trichome count. These groups will make up the two artificially selected lines: high hairiness and low hairiness, respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If more than 10% of the plants have no trichomes, select 10% of those plants randomly for the low hairiness plant line.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Separate the two groups of selected plants and label them clearly.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Move the plants to a well-lit area and keep them well watered until they develop flowers. Note: Flowers typically develop around 14 days.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the flowers are fully developed, use a pollination wand or paintbrush to rub the stigma and anther of one plant&#8217;s flowers for a few seconds.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using the same wand or brush, rub the inside of each flower of every other plant in the same selection line.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using a different wand or brush, repeat steps 11 through 12 on the other line. Note: Take care not to cross the lines. Spread the pollen only among the appropriately selected plants.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Return the generation one plants to the growing areas. When the plants reach approximately 1 month old, they should produce mature seed pods.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the plants to dry out once the seed pods turn yellow.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Break the seed pods open and collect the seeds.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Obtain new pots for the generation two seeds.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Fill the pots with moist potting soil about halfway and add a fertilizer pellet to each pot.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Fill the pots with more soil to fill just below the lip.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Make a shallow depression in the soil of each pot with a probe.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Touch a clean toothpick to a gluestick and use the toothpick to transplant the seeds into the soil one by one.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Cover the seeds with a thin layer of soil.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Label the pots with their hairiness line and place them in the designated growing area.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Use a plastic dropper to water the plants as needed to ensure they do not dry out.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">When the plants are two weeks old, collect five plants from each line and label them A to E.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, count the trichomes on these plants as previously described (steps 4- 5).
<a target="__blank" style="display:block;padding:20px;" href="https://www.jove.com/CDNSource/lm/labs/2/Lab_2_Table_B.xlsx">Click Here to download Table B</a>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Create a frequency graph based on the generation one class data with the trichome count on the x axis and the number of plants on the y axis. Label the mean. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, create frequency graphs for both of the selected hairiness lines.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare the frequency graphs for generation one and both hairiness lines.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Take note of phenotypic shifts observed in the selected hairiness lines.
</div></li></ul></li></ol>

Artificial Selection: Visualization in Plants and Data Analysis - Procedure

Seleção artificial

Observing Artificial Selection in Plants 
ExpandTo begin the experiment, collect five two-week old grown plants from the generation one population that ...

Evolução

Natural Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Natural Selection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that are 3 inches long.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, cut the green pipe cleaners in the quantities and lengths specified in the video, taking care to minimize waste. This approximates a normal distribution of lengths and should result in an additional 100 pieces total. NOTE: One 12-inch pipe cleaner should yield multiple lengths, for example, a 5-inch piece and a 7-inch piece. Use this advantage to reduce waste.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Now, cut one sheet of black and one sheet of white construction paper into approximately 3-inch strips measured along the long edge of the paper.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Tape these strips down to cover the base of a dissecting tray to create an alternating pattern of black and white strips. NOTE: In the first simulation, you will use the white, gray, and black pipe cleaner segments against a black and white background to simulate different selection pressures due to predation on a population. You will select your prey using two different strategies, eyes-open and eyes-closed. For each of these strategies, you will perform four rounds consisting of 25 selections, alternating between two partners. After each round you will restore the population back to its original size using the proportions of phenotypes present after selection.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To begin the first scenario, count out 12 white, 12 black, and 26 gray pipe cleaner segments with your partner. Hypotheses: Here the experimental hypothesis might be that different predation strategies, such as having the eyes open or closed, will result in distinct selection pressures on the populations. Further, different phenotypes may be positively selected in the alternate scenarios. The null hypothesis might be that different predation strategies will have no effect on the populations or phenotypes.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Mix the pipe cleaner segments together as best as possible. Then, sprinkle them into the papered tray. NOTE: Ideally, the pipe cleaners will be spread out. However, this may not occur due to their self-adhering nature.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once the segments are in the tray, separate them as best as possible without moving them too far from where they landed. As few segments as possible should be touching.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Have the first partner close their eyes. When given an audible signal by the second partner, the first should open their eyes and grab the first pipe cleaner they see from the tray as quickly as possible.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Set the removed pipe cleaner to the side.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Alternating partners, repeat this process a total of 25 times (steps 8 &#8211; 9).
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Count the remaining pipe cleaners by color and record this in the data collection table for Scenario 1. <a href="https://www.jove.com/CDNSource/lm/labs/23/Lab_23_Tables_1_and_2.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 1 and 2</a>
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, restore the population to 50 by adding one pipe cleaner of the same color for each of the remaining pipe cleaners. Hold these to the side until they are all gathered.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then, sprinkle the pipe cleaners into the tray as previously described (steps 6-7). Repeat this process for three more rounds consisting of 25 selections each and record the counts for each of the rounds in the data collection table for Scenario 1.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Remove all pipe cleaners from the tray after the fourth round.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For Scenario 2, again count out 12 white, 12 black, and 26 gray pipe cleaner segments and sprinkle them randomly into the tray.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Follow the same protocol as in Scenario 1. However, when given the signal, the grabbing partner should keep their eyes closed. To aid in accuracy, it may help to position the hand near to the tray before grabbing.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Repeat this process 25 times, switching partner roles, and record the remaining pipe cleaner amounts in the data collection table for Scenario 2.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, restore the population to 50 based on that count. Do this for all four rounds. NOTE: In the second simulation, you will be using the eyes-open and eyes-closed predation strategies as previously described to select pipe cleaners of various lengths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">For Scenario 3, gather all 100 of the pipe cleaners that have been cut to varying lengths to represent different phenotypes.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">One partner should hold all the pipe cleaners upright and loosely in one hand resting on the tabletop, such that the bottom end of each pipe cleaner rests against the tabletop.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, the partner that is not holding the pipe cleaners should close their eyes. When given an audible signal, the partner should open their eyes and grab the first pipe cleaner they see from their partner&#39;s hand.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process 50 times (step 21). However, the partners should not switch roles during the round this time. The partner holding the pipe cleaners should always be the one giving the signal and never grabbing from the bunch.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">After the first round consisting of 50 selections, count the remaining pipe cleaners by length. Then, record this information in the data collection table for scenario three. <a href="https://www.jove.com/CDNSource/lm/labs/23/Lab_23_Tables_3_and_4.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 3 and 4</a>
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Add one pipe cleaner of the same length as each of the remaining ones to restore the population to 100. This will likely require more pipe cleaners than were originally cut. Try to use scraps from the class to reduce waste.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Repeat this for three more rounds, alternating which partner holds or grabs each round.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">To perform Scenario 4, gather the varied-length pipe cleaners in their original proportions.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Now repeat the grabbing process, as described in Scenario 3. However, only remove 30 pipe cleaners instead of 50.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Divide the count at the end of each round by two for each phenotype 4 inches or below, rounding fractions down.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Then, record these values in the data recording table for Scenario 4.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Use the relative proportions of the phenotypes in the final population to restore the population to 100. When restoring the population to 100, it may be necessary to calculate relative proportions of phenotypes.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Repeat this for three more rounds, again alternating holding and grabbing the pipe cleaners.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">For each scenario calculate the survival for each phenotype, that is the proportion of the phenotype surviving.
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Then, calculate the relative fitness, or w, between each round. As reproductive rate does not vary in this simulation, this should always be considered equal, or 1.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Also, calculate the selection coefficients for each phenotype.
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Now, use the relative frequencies of each phenotype to create histograms with appropriately labeled axes for each scenario showing the population before any selection took place and then alongside these in a different color after four rounds of selection.
</div></li><li data-sub-note-item="1-36"><div class="lab-step">Identify the type of selection taking place in each scenario and label the graphs. 
</div></li><li data-sub-note-item="1-37"><div class="lab-step">Develop a hypothesis to explain your observations. 
</div></li></ul></li></ol>

Natural Selection: Calculating Fitness and Selection - Procedure

Seleção natural

Simulating Natural Selection
ExpandTo begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that ...