Name: laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates
Uploaded: 2017-05-30T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

研究由新罕布什尔州INBRE通过机构发展奖（IDeA）支持，P20GM103506来自美国国家卫生研究院国家综合医学研究所。

注意:每个测定的时间考虑必须作为单个培养基制备需要不同的时间。例如，应将固体琼脂平板培养基充分冷却并干燥（1-2天）。额外的时间考虑（ 即单菌落和隔夜培养生长）在每个方案下规定， 见表2 。 媒体准备<ol><li> 1x磷酸缓冲盐水（PBS） <ol><li>称取4.0g NaCl，0.1g KCl，0.72g Na 2 HPO 4和0.12g KH 2 PO 4 。将组分在1升锥形烧瓶中，用去离子水将体积调节至500mL，使用pH计将pH调节至7.2，并将HCl滴加到溶液中，并使用0.22μm过滤器进行高压灭菌或过滤灭菌。 1x PBS可以无限期地在室温下储存。 注意:HCl是浓缩酸，应按照以下方式处理到制度，地方，州和联邦法规。对于适当的处理指南，请参阅制造商提供的安全数据表。 </li></ol></li><li> Luria-Bertani（LB）肉汤 <ol><li>称重5.0g胰蛋白胨，2.5g酵母提取物和2.5g NaCl。将组分在1升瓶中，用去离子水调节体积至500mL，高压灭菌，并在室温下储存长达6个月。对于经典的生物型毒力诱导条件，在高压灭菌前用HCl调节pH至6.5。 </li></ol></li><li> 含有0.03％（w / v）NaHCO 3的 AKI培养基 <ol><li>对于组分1（AKI培养基）:重7.5g蛋白胨，2.0g酵母提取物和2.5g NaCl。将组分在1升瓶中，用去离子水和高压釜将体积调节至450mL。 AKI培养基可以在室温下储存长达6个月。 </li><li>对于组分2（NaHCO 3 ）:称重1.5g NaHCO 3 ，并调节体积在无菌囊泡中用去离子水加至50mL。使用0.22μm过滤器过滤灭菌NaHCO 3 。必须在使用前立即制备NaHCO 3 。 </li><li>在使用前将组分2过滤到组分1中，无菌地组合两种成分，产生1:10的比例。 </li></ol></li><li> Luria-Bertani（LB）琼脂板 <ol><li>称取5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和7.5g琼脂。将组分在1升锥形烧瓶中，用去离子水，高压釜调节体积至500毫升，并在标准尺寸的100毫米×15毫米无菌培养皿中制备。电镀介质可以保存在塑料中长达6个月，盖子朝上放置在4°C。 </li></ol></li><li> LB琼脂平板补充多粘菌素B <ol><li>对于组分1（LB琼脂）:称重5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和7.5g琼脂。将组分在1升锥形瓶中，将体积调节至500mL wi去离子水和高压釜。 </li><li>对于组分2（多粘菌素B）:将无菌2mL微量离心管中的去离子水中的多粘菌素B溶解至50,000IU /μL的浓度，并使用0.22μm过滤器进行过滤消毒。 </li><li>一旦组分1冷却，但尚未固化，无菌地向组分1中加入500μL组分2，产生最终浓度为50IU /μL并充分混合。通过将混合的琼脂培养基倒入培养皿中制备标准尺寸的100mm×15mm无菌培养皿。电镀介质可以保存在塑料中长达3个月，盖子在4℃升高。 </li></ol></li><li> 柠檬酸盐中等琼脂平板 <ol><li>对于组分1（具有溴百里酚蓝的琼脂）:称重7.5g琼脂和0.02g溴百里酚蓝。将组分在1升三角烧瓶中合并，用去离子水将体积调节至450mL，并将混合物高压。 </li><li>对于组件2（10x VBMM）:称重10g MgSO 4 7H 2 O，10.0g柠檬酸H 2 O，50.0g无水K 2 HPO 4和17.5g NaNH 4 HPO 4 4H 2 O。将组分在1升锥形瓶中，将体积调节至500mL用去离子水和高压灭菌器或使用0.22μm过滤器进行过滤消毒。 10x VBMM可在室温下储存6个月。 </li><li>通过将混合的琼脂培养基倒入培养皿中，将50 mL组分2加入到组分1中，并制成标准尺寸的100 mm x 15 mm无菌培养皿。电镀介质可以保存在塑料中长达6个月，盖子朝上放置在4°C。 </li></ol></li><li> 牛奶琼脂板 <ol><li>对于组分1（牛奶）:在1L三角烧瓶中称重8.0g速溶脱脂乳，用去离子水和高压釜调节体积至200mL。 </li><li>对于组分2（具有脑心脏输注的琼脂）:称重3.68g脑 - 心脏输注和6.0g琼脂。结合con在500mL三角烧瓶中的成分，用去离子水和高压釜将体积调节至200mL。 </li><li>通过将组分2倒入组分1中，高压灭菌后彻底混合，合并两种组分。在150毫米x 15毫米无菌培养皿中准备。牛奶板可以储存长达一周，塑料包装，盖子侧面在4°C下方。通过将混合的琼脂培养基倒入培养皿中制备150毫米×15毫米无菌培养皿。 </li></ol></li><li> 动力琼脂板 <ol><li>对于组分1（LB琼脂）:称重5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和2.0g琼脂。将组分在1升锥形瓶中合并，用去离子水和高压釜调节体积至500mL。 </li><li>对于组分2（1％（w / v）三苯基四唑氯化物; TTC）:称重0.25g TTC。在无菌囊泡中用去离子水将体积调节至25 mL，并使用0.22μm过滤器进行过滤消毒。存放长达6个月的室温包裹在箔中以最小化曝光。 </li><li>加入2.5 mL 1％（w / v）无菌TTC至高压灭菌培养基。 </li><li>将介质尽可能厚（≥50mL /平板）倒入大型150 mm x 15 mm无菌培养皿中。电镀介质可以保存在塑料中长达一周，盖子在4℃升高。通过将混合的琼脂培养基倒入培养皿中制备150毫米×15毫米无菌培养皿。 </li></ol></li><li> Voges-Proskauer（副总裁） <ol><li>对于组分1（甲基红Voges-Proskauer肉汤; MR-VP）:在500mL锥形瓶中重1.7g MR-VP培养基，用去离子水和高压釜调节体积至100mL。无菌MR-VP肉汤可以在室温下储存长达6个月。 </li><li>对于组分2（5％（w / v）α（α）萘酚）:在无菌囊泡中称重1.0gα-萘酚，并用95％乙醇调节体积至20mL。 α-萘酚可以在室温下储存长达1周的箔包裹，以使光透过率最小化osure。 </li><li>对于组分3（40％（w / v）氢氧化钾; KOH）:在无菌泡囊中重量为20.0g KOH，用无菌去离子水将体积调节至50mL。 KOH可以在室温下在塑料容器中储存多达6个月。 </li></ol></li></ol>维持和增长V。 霍乱弧菌菌株<ol><li> 冷冻库存培养的准备和使用 <ol><li>颗粒将1.8mL液体过夜培养2分钟，在无菌2mL微量离心管中离心（≥8,600xg），除去上清液，并通过移液将细胞沉淀重新悬浮在900μL新鲜LB肉汤中。 </li><li>向培养基中加入900μL无菌60％（v / v）甘油，并通过涡旋混合。 </li><li>将混合物转移到无菌2 mL低温管中，无限期储存在-80°C。 </li><li>为了使用，使用无菌接种环和少量冷冻原料，在LB ag上用于单个菌落ar板。使用后立即将冷藏库存回到-80°C，以防止文化完全解冻。将盖板盖在37℃下孵育12至16小时。 </li></ol></li><li> 液体培养基中过夜培养物的生长 <ol><li>将单个菌落（根据2.1.4节）从冷冻原料连续到LB琼脂平板上。将盖板盖在37℃下孵育12至16小时。 </li><li>通过用无菌涂抹棒接触单个菌落的表面，将4mL液体LB肉汤接种在具有单个菌落的无菌10mL培养管中，并将菌落转移到液体肉汤中。 </li><li>在振荡器培养箱中以225rpm的通气温度在37℃下孵育12至16小时。 </li></ol></li><li> 增长曲线 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>通过将250μL过夜培养物转移到2，进行1:100稀释的过夜培养将5mL新鲜LB肉汤在无菌的250mL锥形瓶中。 </li><li>在接种时开始每小时测量600nm（OD 600 ）的液体培养物的光密度（T 0 ）。 </li><li>孵育1:100稀释的过夜培养物，在振荡器培养箱中以225rpm通气37℃培养达30小时，或培养达到最大密度。 </li><li>将OD 600与时间对照，并使用线性回归确定每个菌株的近似加倍时间。 </li></ol></li><li> El Tor生物型毒力诱导条件 <ol><li>将单个菌落从冷冻原液连续到LB琼脂平板上。将盖子盖板向下孵育，在37℃下放置12至16小时。 </li><li>接种含有0.03％（w / v）NaHCO 3的 10mL含有单个菌落的AKI培养基。 </li><li>孵育培养物，37℃不通气3.5小时。 </li><li>取出7 mL培养液，孵育剩余的3 mL培养液在振荡器培养箱中以225rpm曝气另外4小时。 </li></ol></li><li> 古典生物型毒力诱导条件 <ol><li>将单个菌落从冷冻原液连续到LB琼脂平板上。将盖板盖在37℃下孵育12至16小时。 </li><li>用单个菌落接种4mL液体LB肉汤（pH 6.5）。 </li><li>在振荡器培养箱中以225rpm在30℃下通气曝气12至16小时。 </li></ol></li><li> 在1x PBS中洗涤细胞颗粒 <ol><li>在无菌的2mL微量离心管中通过离心（≥8,600xg）将1.8mL过夜培养物沉淀2mL，并用移液管除去上清液。通过移液将细胞沉淀重悬于1.8 mL 1x PBS。 </li><li>重复洗涤程序三次，最后重悬于1.8 mL 1x PBS中。 </li></ol></li></ol>表征V。 霍乱生物型<ol><li> P使用ctxB和tcpA的基于CR的遗传筛选<ol><li>设计一组引物，分别在ctxB和tcpA的翻译启动和停止位点的上游和下游退火约50-70bp。 </li><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>使用为革兰氏阴性菌设计的市售试剂盒分离染色体DNA。纯化的染色体DNA可以无限期储存在-20°C。使用市售试剂盒的染色体DNA分离通常需要约4小时。 </li><li>使用微量分光光度计确保染色体DNA样品质量高（A 260/280 &gt; 1.8）。 </li><li>对于每个染色体DNA分离物，在无菌200μLPCR管中在冰上制备聚合酶链式反应（PCR），并使用标准PCR组分扩增ctxB和tcpA的区域: Taq聚合酶和buffer（或等同物），dNTP溶液和正向/反向引物。使用标准PCR参数（〜3 h）。例如，使用以下协议: <ol><li> 95°C初始变性120 s。 </li><li>在95℃变性60秒。 </li><li>退火底漆在60℃45秒。 </li><li>在72℃延伸90秒。 </li><li>重复步骤3.1.5.2至3.1.5.4进行34个周期。 </li><li>最终延伸72°C，持续600 s。 </li><li>无限保持在4°C。 </li></ol></li><li>使用标准的6x DNA凝胶加载染料染色5μL各自的PCR产物，并将大约10μL的1kb梯度和10μLPCR产物加载到1×Tris-硼酸盐-EDTA（TBE）中的1％琼脂糖凝胶上，缓冲。 </li><li>在130 V下运行凝胶电泳，直到染料前端达到凝胶的末端，但不能脱离凝胶（约90分钟）。建议进行恒定监控，因为电压和运行时间因设备而异。 </li><li>经验证以预期的大小成功进行PCR扩增，使用市售的DNA清洁和浓缩器试剂盒纯化剩余的45μLPCR产物。 </li><li>使用与PCR扩增相同的引物进行序列清洗的PCR产物，并根据每个本地测序设备指南进行准备。 </li><li>通过在搜索查询框中搜索以下登录号，将NCAPI网站（http://www.ncbi.nlm.nih.gov/gene/）上可获得的每个分离株的基因序列的FASTA格式与已公布的序列进行比较。 </li><li> ctxB :VC0395_A1059到VC0395_A1060（El Tor）VC_1456之间的（经典）区域</li><li> tcpA（经典）VC0395_A0353（El Tor）VC_0828 </li></ol></li><li> 通过斑点生物型分类的表型分析 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>洗涤方案2.6中规定的细胞沉淀。 </li><li>使用移液器在各自的培养基上洗涤1μL洗涤的培养物。有关中等选择和孵化规格，请参见表2。 </li></ol></li><li> 动力测定 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养物。 </li><li>洗涤方案2.6中规定的细胞沉淀。 </li><li>通过将接种刺入插入洗涤的液体培养物中并"垂直地"插入培养基中来接种动力琼脂平板。在每次接种之间，使用本生灯灭菌线刺，并且当刺入琼脂时，确保接种刺不弯曲，因为这样可以改变结果。 </li><li>将盖板盖在37℃下孵育14至24小时。 14小时后，密切监测动力板，以防止文化过度生长。 </li></ol></li><li> Voges-Proskauer（VP）测定 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养物。 </li><li>接种4mL的甲基红Voges-Proskauer或MR-VP肉汤，通过在无菌培养管中将10μL先前制备的过夜培养液移液到4mL MR-VP肉汤中，并在振荡器培养箱中以225rpm通气12至16℃ h在37°C。 </li><li>在无菌培养管中分别加入150μL5％（w / v）α-萘酚和50μL40％（w / v）KOH至1mL等分的MR-VP过夜培养。 </li><li>短暂旋转并在室温下放置4小时，直到发生变色。 </li></ol></li></ol>

<ol>
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作者没有什么可以披露的。

在200多个确定的V。 霍乱血清群，只有O1和O139具有流行潜力。 O1血清群可以分为两种生物型:古典和El Tor。然而，已经出现了称为El Tor变体13,17的杂交菌株，具有El Tor生物型背景，并具有古典特征8,9,10,11,12,13,14,15,16,17。本手册中描述的方案旨在提供有兴趣表征和/或区分霍乱弧菌的各种临床和非临床分离株的研究者，具有可靠的多检测鉴定系统。使用可靠的多分析鉴定系统作为替代方案是对先前建立的单一测定鉴定系统和劳动密集型遗传筛选的改进。所有基因型和表型测定应包括WT经典菌株O395和WT El Tor菌株C6706和N16961进行比较（ 表1 ）。虽然包括古典和El Tor生物型菌株供参考，但我们研究中包括的分离株如MQ1795和BAA-2163可以显示来自生物型的表型谱（ 图2 ， 图3 ， 图4 ， 图5 ， 图6 ， 图7 ; 表2 ），说明需要分析多种基因型和表型性状以进行可靠的表征。所有协议应无菌进行除非另有说明，否则室温下为26 。 V。 霍乱弧菌是一种生物安全二级（BSL-2）病原体，它是致命性胃肠道疾病霍乱的病原体;必须根据制度，地方，州和联邦法规对所有材料和废物进行适当处理和处置。 所有培养基和试剂的制备必须使用分析纯试剂和超纯水去离子水进行，最小灵敏度为18MΩ-cm。加工前，应准备培养基使其充分干燥（1-2天）;当琼脂表面上没有残留的液体时，板被认为是足够干燥的。由于围绕细菌生长的活动范围和间隙区域，运动性和奶板应在每平板最多50 mL的大培养皿（150 mm x 15 mm）内制备，并可储存长达1周的塑料包裹，李d侧向下4℃。另外，运动板的琼脂浓度可以调节以减缓运动;然而，不建议每500 mL溶液超过3 g琼脂，因为这将大大降低总运动性，从而不能观察到差异。所有其他平板培养基应准备在标准尺寸的培养皿（100mm×15mm）中每平板约25mL，并且可以储存长达六个月的塑料，盖子侧向下放置在4℃。对于多粘菌素B板的制备，在加入多粘菌素B之前，在高压灭菌后允许熔融介质冷却是重要的，因为过量的热量可能降解抗生素。多粘菌素B板可以储存长达3个月，塑料包装，盖子在4℃下降。在本手稿中讨论的测定需要一天的单一菌落生长（12-16小时），另外一天用于培养过夜培养（12-16小时），第三天考虑基因型（染色体DN约4小时）分离和PCR约3小时）或表型测定（18-48小时; 表2 ）。在本手稿中描述的表型测定的生物化学分析之前，培养物应在1x PBS中洗涤，以防止残留培养基携带偏斜结果。此外，多粘菌素B，柠檬酸盐，蛋白酶活性，溶血和运动性测定可以使用单次洗涤的过夜培养物同时接种。当发现电镀培养基时，可能会发生飞溅的培养物，并可能导致菌株之间的交叉污染。为了防止飞溅，避免从移液器中完全喷出培养物，而应停止在移液器第一个停止处喷出培养物。另外，在孵育前让斑点充分吸收到琼脂表面，注意不刺穿琼脂，这可能会影响结果。表型测定导致清除区（ 图4 ; 图5 ; 表2）和/或运动性（ 图6 ; 表2 ）应最大限度地间隔开，以防止分别可以测量各自的直径以进行比较分析的间隙或生长区域的合并。在指示的孵育时间后，可以相对于生物型参考菌株（WT经典O395，WT El Tor C6706和WT El Tor N16961）成像和分析分离物。 在初步PCR扩增后，本手稿中概述的基于PCR的遗传筛选可用于鉴定相对于ctxB和tcpA的分离株的生物型背景。标准测序指南需要特定量的PCR产物，取决于扩增区的大小（ ctxB为580bp，tcpA为1420bp），并且为了建立反应，可以遵循建立的方案。简单来说，个人前进和r应用3-5 pmol引物/反应制备反向测序反应，用无菌超纯水在1.5 mL微量离心管中调节体积至20μl。为了帮助PCR扩增和测序的引物设计，可以使用NCBI引物设计工具http://www.ncbi.nlm.nih.gov/tools/primer-blast/。使用以下引物（5&#39;→3&#39;）成功扩增和测序ctxB和tcpA: ctxB-正向GGGAATGCTCCAAGATCATCGATGAGTAATAC， ctxB-反向CATCATCGAACCACAAAAAAGCTTACTGAGG，tcpA -forward CCGCACCAGATCCACGTAGGTGGG，tcpA-逆GTCGGTACATCACCTGCTGTGGGGGCAG。应该注意的是， ctxB的整个编码区域除了基本位置115和203（El Tor中的经典和胸腺嘧啶中的胞嘧啶）之外的两种生物型都是保守的。此外，在tcpA中，多重碱基变化在d的生物型之间保守如果穿过两种生物型（ 表1 ），可用于帮助区分生物型。 在涉及模型生物体V的许多实验室研究中。 霍乱 ，适当的维护和培养技术至关重要27 。常用V增长率霍乱生物型参考菌株，如WT经典菌株O395和WT El Tor菌株N16961，可以为El Tor变异株的表征提供有用的见解（ 图1 ; 表2 ）。由于V的增长速度不同。 霍乱弧菌分离物，重要的是每小时取分光光度计的吸光度读数，直到培养物在晚期固定期达到最大浊度。由于多余的细胞碎片造成的浊度平缓，中晚期死亡期可能无法显现。 1:4稀释培养至无菌L<html>应该执行B肉汤以获得完整的生长曲线并保持仪器的精度，因为吸光度读数在OD 600≈1.0时达到峰值。当在多个菌株上进行生长曲线时，每个菌株的吸光度读数应该相隔大约5分钟，以确保读数之间的一致性。了解V 霍乱生物型生长速率对许多研究至关重要，包括毒力基因表达研究，代谢活动分析和适当的培养和储存条件。对于随后的分析，应将晚间培养物加工成早期稳定生长期（12-16小时），以使细胞生长最大化，同时保持细胞完整性。 经典和El Tor生物型菌株的培养条件是相似的，然而， V中毒力基因表达的分析。 霍乱需要生物型特异性毒力诱导条件参考文献19。两种主要毒力因子CT和TCP由主调节剂ToxT控制，并在特定生长条件下进行最佳表达;例如，在El Tor毒力诱导条件下，ToxT可以通过加工全细胞提取物WCE）或细胞沉淀，3.5 h时，CT和TCP表达可以分别在7.5 h处理无细胞上清液和WCE分别为8,20 ，本手稿中显示的不同基因型和表型性状如何不同的霍乱弧菌生物型可以是，并且说明需要替代先前使用的单一测定生物型表征。

霍乱是由食用含有水生革兰氏阴性菌霍乱弧菌的污染食物或水引起的远端小肠疾病。 霍乱的症状包括呕吐和不可控制的水样腹泻，导致严重脱水，如果不妥善治疗，将导致死亡。 V。基于细胞表面脂多糖O-抗原的结构， 霍乱弧菌可以分为200多个血清群。然而，只有2个血清群，O1和O139，已显示出流行或大流行的潜力1,2 。此外，血清群O139主要分离到东南亚3,4 ，而血清群O1在全球分布。此外，O1血清群可以分为两种主要生物型:古典和El Tor。经典生物型是造成头6头霍乱流行病1817年和1923年之间。正在进行的第七大流行病是El Tor生物型的结果，全球在环境5,6,7的环境中使古典生物型流行病学变异。最近，出现了具有古典和El Tor生物型8,​​9,10,11,12,13,14,15,16,17的区别特征的菌株，并且之后被称为El Tor变体13,17。一些El Tor变体已经显示出比以前观察到的更迅速和严重的疾病进展的升高的毒力能力，强调需要一个更全面的方法来代理身份识别和疾病预防和治疗8,9,18。虽然生物型鉴定并不立即决定治疗，但疫苗开发和未来治疗剂的进一步进步可能受益于生物型别的区别。 这里列出的第一系列协议将使调查人员能够正确维护V。 霍乱弧菌菌株在实验室设置。一致性和随后的分析需要分离物的储备准备和生长，其不是生物型依赖性的。然而，为了最佳地诱导毒力基因表达，需要独立的生物特异性培养技术。此外，本手稿中概述了各种遗传和生物化学测定的准备工作。 霍乱毒素（CT）和毒素共同作用培养的真菌（TCP）是主要调节剂ToxT在V的两种生物型中控制的两种主要的毒力因子。 霍乱弧菌 O1血清群20 。 CT是由五个CtxB组成的两部分毒素 亚单位围绕单个CtxA亚单位，并且负责与霍乱相关的快速电解质损失。 TCP是由tcp操纵子（ tcpABQCRDSTEF ）编码的IV型pilus ，并且涉及远端小肠的附着和定殖。 tcpA是tcp操纵子的第一个基因，其编码构建真菌8所必需的各个pilin亚单位。 ctxA的基因序列在经典和El Tor生物型之间是完全保守的，而ctxB和tcpA在两种生物型之间不同，但在每种生物型8中都是保守的。除了两个基本位置之外， ctxB在生物型之间是完全保守的（115和203）。在El Tor生物型中，胸腺嘧啶位于基因位置115和203，而经典生物型在这些碱基处含有胞嘧啶。 tcpA在每种生物型中都是完全保守的，但在生物型之间的多个基础上是不同的。这些遗传差异用作初级生物型鉴定标记，并且在测序包括这些位点的聚合酶链反应（PCR）扩增产物之后，可以将分离序列与野生型（WT）经典O395或WT E1 Tor N16961进行比较，以确定生物型背景的CT和TCP分别在给定的霍乱弧菌分离株中。 已经开发了许多方案来表征经典和El Tor生物型21,22,23之间的表型区别。多粘菌素B是一种肽抗生素，其在革兰氏阴性杆菌中损害外细胞膜的完整性通过多粘菌素B抗性测定可以观察到teria和多粘菌素B抗性21 。柠檬酸盐是克雷伯氏循环的主要底物，并且可以使用柠檬酸盐代谢测定法22来确定将柠檬酸盐代谢为唯一碳源的能力。 hapR编码霍乱弧菌 HapR中的全局调节子和主要群体感应调节因子，其结合各种启动子区域并调节基因和操纵子表达24 。 霍乱弧菌的一些致病菌株在hapR基因中具有天然存在的转录突变，导致毒力基因表达的密度依赖性调节丧失24,25 。使用牛奶琼脂培养基测量HapR调节的蛋白酶活性允许研究者鉴定特定分离物是否含有功能性HapR 23 。该溶血分析测试菌株分泌溶血红细胞的溶血酶的能力;溶血程度可以在血琼脂平板23上显现。运动性通常与霍乱弧菌中的毒力相关，可以使用运动性琼脂平板23进行分析。 Voges-Proskauer测定法测试菌株作为唯一碳源发酵葡萄糖并产生副产物乙偶姻21的能力 。随着El Tor变体的出现，很难预测任何给定的表型测定的结果，而没有广泛的基因型筛选，并且在推导V的生物型背景之前。 霍乱弧菌分离物，推荐进行这种测定23的装配，并将结果与表2中的参考菌株进行比较。 在这里，我们提出了一系列方案，集体利用上述方案基因型和表型检测用于更全面的表征V的方法 。 霍乱生物型此外，我们已经描述了已知V的基因型和表型区别。与常用的生物型参考菌株（WT经典O395，WT E1 Tor C6706和WT El Tor N16961; 表1 ）相比， 霍乱弧菌 El Tor变体（MQ1795和BAA-2163）。 El Tor变体的出现对先前使用的单一测定生物型表征方案的可靠性提出了挑战;然而，这种多重测定鉴定系统将允许更可靠地表征临床和环境V。 霍乱弧菌分离株

<table><tbody><tr><td>1 kb DNA Ladder</td><td>New England Biolabs</td><td>N3232S</td><td>https://www.neb.com/products/n3232-1-kb-dna-ladder</td></tr><tr><td>60% Glycerol</td><td>Calbiochem</td><td>356352</td><td>http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352</td></tr><tr><td>Agar</td><td>Becto, Dickinson and Co.</td><td>214030</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch</td></tr><tr><td>Agar with brain-heart infusion</td><td>Becto, Dickinson and Co.</td><td>237500</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch</td></tr><tr><td>Agarose</td><td>Peqlab</td><td>732-2789</td><td>https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&amp;amp;_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&amp;amp;_dynSessConf=1766917479792147141&amp;amp;targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&amp;amp;lastLanguage=en&amp;amp;/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&amp;amp;_D%3AcurrentLanguage=+&amp;amp;currentLanguage=en&amp;amp;_D%3AlastLanguage=+&amp;amp;_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+</td></tr><tr><td>Anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P288-500</td><td>https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&amp;amp;searchTerm=P288500&amp;amp;searchType=RAPID</td></tr><tr><td>Blood Agar Plates</td><td>Remel</td><td>R01200</td><td>Store at 4 &amp;deg;C;&amp;nbsp; http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar</td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>A73-500</td><td>https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&amp;amp;searchTerm=A73500&amp;amp;searchType=RAPID</td></tr><tr><td>Bromothymol Blue</td><td>Fisher Scientific</td><td>B388-10</td><td>https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&amp;amp;searchTerm=B38810&amp;amp;searchType=RAPID</td></tr><tr><td>Cirtric acid &amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S72836-3</td><td>https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363</td></tr><tr><td>Deoxynucleotide (dNTP) Solution Kit</td><td>New England Biolabs</td><td>N0446S</td><td>Store at -20 &amp;deg;C;&amp;nbsp; https://www.neb.com/products/n0446-deoxynucleotide-solutionset</td></tr><tr><td>Disodium EDTA</td><td>Fisher Scientific</td><td>S311-500</td><td>https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&amp;amp;searchTerm=S311500&amp;amp;searchType=RAPID</td></tr><tr><td>DNA Clean &amp;amp; Concentrator&amp;trade; -25 Kit</td><td>Zymo Research</td><td>D4007</td><td>http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit</td></tr><tr><td>GelGreen Nucleic Acid Stain</td><td>Biotium</td><td>41005</td><td>https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10,000x-in-water/</td></tr><tr><td>Genesys 10SUV-VIS Spectrophotometer</td><td>Thermo Scientific</td><td>840-208100</td><td>https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100</td></tr><tr><td>Gentra Puregene Yeast/Bact. Kit</td><td>Qiagen</td><td>158567</td><td>https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation</td></tr><tr><td>HCl</td><td>Fisher Scientific</td><td>A144-212</td><td>Corrosive;&amp;nbsp; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&amp;amp;searchTerm=A144212&amp;amp;searchType=RAPID</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217-500</td><td>https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&amp;amp;searchTerm=P217500&amp;amp;searchType=RAPID</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td>https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&amp;amp;searchTerm=P285500&amp;amp;searchType=RAPID</td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td>P250-500</td><td>https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&amp;amp;searchTerm=P250500&amp;amp;searchType=RAPID</td></tr><tr><td>Le Loop</td><td>Decon Labs Inc.</td><td>MP190-25</td><td>http://deconlabs.com/products/leloop/</td></tr><tr><td>Le Stab</td><td>Decon Labs Inc.</td><td>MP186-5</td><td>http://deconlabs.com/products/lestab/</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>M63-500</td><td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&amp;amp;searchTerm=M63500&amp;amp;searchType=RAPID</td></tr><tr><td>Mini-Sub Cell GT Horizontal Electrophoresis System</td><td>Bio Rad Labs</td><td>1704406</td><td>http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&amp;amp;WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&amp;amp;WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373</td></tr><tr><td>MR-VP Broth</td><td>Difco</td><td>216300</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td>https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&amp;amp;searchTerm=S374500&amp;amp;searchType=RAPID</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-10</td><td>https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&amp;amp;searchTerm=S27110&amp;amp;searchType=RAPID</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-500</td><td>https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&amp;amp;searchTerm=S233500&amp;amp;searchType=RAPID</td></tr><tr><td>NaNH&lt;sub&gt;4&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S218-500</td><td>https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&amp;amp;searchTerm=S218500&amp;amp;searchType=RAPID</td></tr><tr><td>NanoDrop Lite Spectrophtometer</td><td>Thermo Scientific</td><td>ND-LITE-PR</td><td>https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR</td></tr><tr><td>Nonfat dry milk</td><td>Nestle Carnation</td><td>N/A</td><td>N/A</td></tr><tr><td>Peptone</td><td>Becto, Dickinson and Co.</td><td>211677</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch</td></tr><tr><td>Petri Dishes (100 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712</td></tr><tr><td>Petri Dishes (150 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875714</td><td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&amp;amp;searchTerm=FB0875714&amp;amp;searchType=RAPID</td></tr><tr><td>Polymyxin B sulfate salt</td><td>Sigma-Aldrich</td><td>P1004-10MU</td><td>Store at 2-4 &amp;deg;C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&amp;amp;region=US</td></tr><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA Polymerase</td><td>New England Biolabs</td><td>M0273S</td><td>Store at -20 &amp;deg;C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td></tr><tr><td>&lt;em&gt;Taq&lt;/em&gt; Reaction Buffer</td><td>New England Biolabs</td><td>M0273S</td><td>Store at -20 &amp;deg;C;&amp;nbsp; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td></tr><tr><td>Thermal Cycler Bio-Rad C1000 Touch&amp;trade;&amp;nbsp;</td><td>Bio Rad Labs</td><td>1840148</td><td>http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&amp;amp;_pageLabel=search_page&amp;amp;sfMode=search&amp;amp;sfStartNumber=1&amp;amp;clearQR=true&amp;amp;js=1&amp;amp;searchString=1840148&amp;amp;database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&amp;amp;tabName=DIVISIONNAME</td></tr><tr><td>Triphenyltetrazolium chloride</td><td>Alfa Aesar</td><td>A10870</td><td>https://www.alfa.com/en/catalog/A10870/</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&amp;amp;searchTerm=BP1521&amp;amp;searchType=RAPID</td></tr><tr><td>Tryptone</td><td>Becto, Dickinson and Co.</td><td>211705</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch</td></tr><tr><td>Yeast Extract</td><td>Becto, Dickinson and Co.</td><td>212750</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch</td></tr><tr><td>&amp;alpha;-napthol</td><td>MP Biomedicals</td><td>204189</td><td>http://www.mpbio.com/product.php?pid=05204189</td></tr></tbody></table>

laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates

水生革兰氏阴性菌霍乱弧菌是感染性胃肠道霍乱的病原体。由于这种疾病的全球流行和严重程度， V 。 霍乱弧菌已经在环境和实验室环境中得到广泛的研究，需要适当的维护和培养技术。古典和El Tor是组成V的两个主要生物型。 霍乱弧菌 O1血清群，各自显示独特的基因型和表型特征，为生物型表征提供可靠的机制，并需要不同的毒力诱导培养条件。不管任何给定的感染或爆发的致病菌株的生物型是什么，该疾病的标准治疗涉及补充有抗生素方案的补液治疗。然而，生物型分类对于实验室研究可能是必要的，并且可能在生物医学领域具有更广泛的影响。在2000年初，鉴定出了经典和El Tor生物型的基因型和表型性状的临床分离株。新鉴定的杂种，称为El Tor变体，使得临床和环境分离生物型鉴定变得比以前的传统单试验鉴定方案更复杂。除了描述V。 霍乱弧菌一般维护和培养技术，该手稿描述了一系列基于特定基因（ ctxB和tcpA ）基于PCR的遗传筛选和表型测定（多粘菌素B抗性，柠檬酸盐代谢，蛋白水解活性，溶血活性，运动性和葡萄糖代谢通过Voges- Proskauer测定）共同用于表征和/或区分经典和El Tor生物型。一起，这些测定提供了一种有效的系统方法，作为替代使用，或另外用于代表性的昂贵的劳动密集型实验V。 霍乱临床（和环境）分离株。

为了妥善维护和使用任何细菌菌株，建议知道感兴趣的菌株的倍增时间。这里，常用的V的变化的生长速率。通过生长曲线证实霍乱弧菌菌株，并使用线性回归计算近似倍数。 WT El Tor N16961和El Tor变体MQ1795比WT经典O395（〜2 h）显示更短的倍增时间（分别为〜1 h和〜1 h）（ 图1 ; 表2 ）。 霍乱弧菌遗传操作和随后的分析往往依赖于适当区分生物型的能力。基于PCR的遗传筛选和表型测定集体实施为区分V的生物型背景的可靠系统。 霍乱临床和环境我solates;为了表示，包括生物型参考菌株（WT经典O395，WT El Tor C6706和WT El Tor N16961）和代表性的El Tor变体（MQ1795和BAA-2163）（ 表1 ）。 WT经典O395展示了经典的ctxB和tcpA序列。相反，WT El Tor菌株N16961和C6706表现出El Tor ctxB和tcpA序列。有趣的是，MQ1795和BAA-2163含有与O395相当的经典生物型ctxB亚基，但是两个El Tor变体均含有表示El Tor生物型背景的tcpA（ 表1 ）。 WT经典生物型菌株O395显示对多粘菌素B的敏感性，而WT E1 Tor生物型菌株（C6706和N16961）显示抗性并在补充有多粘菌素B的LB琼脂平板上显示出生长。代表性的El Tor变体菌株（MQ1795和BAA-2163）相对于WT El Tor生物型的抗生素类似抗性（C6706和N16961）（ 图2 ; 表2 ）。 WT经典生物型菌株O395在最小柠檬酸盐培养基上不生长，而WT El Tor菌株（C6706和N16961）能够利用柠檬酸盐作为碳源并在最小柠檬酸盐培养基上显示出生长。代表性的El Tor变体生物型菌株（MQ1795和BAA-2163）表现出与WT E1T2生物型菌株（C6706和N16961）相当的生长（ 图3 ; 表2 ）。 WT经典菌株O395和WT El Tor菌株N16961具有非功能性HapR，因此不显示HapR调节的蛋白酶活性; WT El Tor菌株C6706和代表性的El Tor变体（MQ1795和BAA-2163）作为从接种点发出的清除区（ 图4 ; 表2 ）被阳性显示。 WT经典菌株O395不分泌溶血酶，因此eγ溶血，而WT El Tor生物型菌株（C6706和N16961）和代表性的El Tor变体菌株（MQ1795和BAA-2163）分泌溶血酶，其完全溶解接种点周围的红细胞，并显示β-溶血（ 图5 ; 表2 ）。生物型菌株之间和内部的运动不同;然而，与相对较少运动的WT经典菌株O395和WT E1 Tor菌株C6706（ 图6 ; 表2 ）相比，WT El Tor菌株N16961和El Tor变体（MQ1795和BAA-2163）显示出超运动性。 WT经典菌株O395和代表性的El Tor变体不代谢葡萄糖以产生乙偶姻，而WT El Tor生物型菌株产生作为葡萄糖发酵副产物的乙偶姻，如Voges-Proskauer测定中深红色发育所示 （ 图7 ; 表2 ）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 应变 </td><td colspan="3"> ctxB基因 </td><td> 菌毛 </td><td rowspan="2"> 参考 </td></tr><tr><td> 基地115 </td><td> 基地203 </td><td> 生物型 </td><td> 生物型 </td></tr><tr><td> O395 </td><td> C </td><td> C </td><td>古典</td><td>古典</td><td> 8 </td></tr><tr><td> C6706 </td><td> Ť </td><td> Ť </td><td>埃尔托</td><td>埃尔托</td><td> 8 </td></tr><tr><td> N16961 </td><td> Ť </td><td> Ť </td><td>埃尔托</td><td>埃尔托</td><td> 8 </td></tr><tr><td> MQ1795 </td><td> C </td><td> C </td><td>古典</td><td>埃尔托</td><td> 13 </td></tr><tr><td> BAA-2163 </td><td> C </td><td> C </td><td>古典</td><td>埃尔托</td><td> 8 </td></tr></tbody></table> 表1: 霍乱弧菌 参考菌株的 生物型依赖遗传特征 。该表中显示了基因ctxB和tcpA的DNA碱基变化和相对位置。 WT经典O395和WT El Tor菌株N16961和C6706是常用的生物型参考菌株。 MQ1795和BAA-2163是已知的El Tor变体。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 化验 </td><td rowspan="2"> 应用 </td><td rowspan="2"> 中等选择 </td><td rowspan="2"> 孵化 </td><td colspan="5"> 预期成绩 </td></tr><tr><td> O395 </td><td> C6706 </s仲&gt; </td><td> N16961 </td><td> MQ1795 </td><td> BAA-2163 </td></tr><tr><td> 2.3）增长曲线27 </td><td>确定各种霍乱弧菌菌株的倍增时间</td><td> 1.2）液体LB肉汤</td><td>最长30小时（37°C，曝气） </td><td> 〜2 h </td><td> ND * </td><td> 〜1 h </td><td> 〜1 h </td><td> ND * </td></tr><tr><td rowspan="2"> 3.1）使用ctxB和tcpA的PCR基因筛选8 </td><td>区分ctxB的古典和El Tor生物型背景</td><td> N / A </td><td> N / A </td><td>古典</td><td>埃尔托</td><td>埃尔托</td><td>古典</td><td>古典</td></tr><tr><td>区分tcpA的经典和El Tor生物型背景</td><td> N / A </td><td> N / A </td><td>古典</td><td>埃尔托</td><td>埃尔托</td><td>埃尔托</td><td>埃尔托</td></tr><tr><td> 3.2）多粘菌素B抗性21 </td><td>对抗生素多粘菌素B的敏感性</td><td> 1.5）补充有多粘菌素B的LB琼脂平板</td><td> 18 h（37°C） </td><td> - </td><td> + </td><td> + </td><td> + </td><td> + </td></tr><tr><td> 3.2）柠檬酸代谢22 </td><td>能够代谢柠檬酸盐作为唯一的碳源</td><td> 1.6）最小柠檬酸盐培养基琼脂平板</td><td> 24小时（37℃） </td><td> - </td><td> + </td><td> + </td><td> + </td><td> + </td></tr><tr><td> 3.2）酪蛋白水解23 </td><td> HapR调节的蛋白酶活性</td><td> 1.7）牛奶琼脂平板</td><td> 18 h（37°C） </td><td> - </td><td> - </td><td> + </td><td> + </td><td> + </td></tr><tr><td> 3.2）溶血23 </td><td>测量溶血活性</td><td>血琼脂板</td><td> 48 h（37°C） </TD&gt; <td>伽玛</td><td> Beta版</td><td> Beta版</td><td> Beta版</td><td> Beta版</td></tr><tr><td> 3.3）运动23 </td><td>措施运动程度</td><td> 1.8）运动性琼脂板</td><td> 14-24小时（37℃） </td><td> 10毫米</td><td> 15毫米</td><td> 21毫米</td><td> 25毫米</td><td> 29毫米</td></tr><tr><td> 3.4）Voges-Proskauer 21 </td><td>测量发酵glocose并产生乙偶姻作为副产物的能力</td><td> 1.9）液体Voges-Proskauer培养基</td><td>长达4小时（室温） </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td></tr><tr><td colspan="9">注:"ND *"表示未确定; "+"表示正结果; " - "表示负面结果</td></tr></tbody></table> 表2:用于遗传和表型测定的总结 </st荣&gt; 霍乱弧菌 生物型鉴别。本表总结了用于区分本研究中使用的古典和El Tor生物型的各种遗传和表型测定，应用和预期结果。协议号和对特定协议的引用在"Assay"（分析）列中显示。 "ND *"表示未确定; "+"表示正结果; " - "表示负面结果。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55760/55760fig1.jpg" /> 图1: V 。 WT经典O395，WT El Tor N16961和El Tor Variant MQ1795的霍乱生长曲线。通过测量OD 600 e分析生物型参考菌株（WT经典O395和WT El Tor N16961）的生长速率和在37℃通气下在LB肉汤中生长的代表性的El Tor变体MQ1795的生长速率从T 0开始很小时。生长曲线在8个独立的实验重复进行，每个重复代表每次试验的独立培养事件。 WT El Tor菌株N16961和El Tor变体MQ1795相对于WT经典菌株O395（〜2小时）显示较短的倍增时间（分别为〜1小时和〜1小时），如通过较长的倍增时间所观察到的。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55760/55760fig2.jpg" /> 图2:使用补充多粘菌素B的LB琼脂测定多粘菌素B抗性B.通过在补充有50IU /μL多粘菌素B的LB琼脂上生长的能力来测定对抗生素多粘菌素B的抗性。经典菌株O395在琼脂上没有生长增刊与多粘菌素B结合并被认为对抗生素敏感。虽然WT El Tor菌株（C6706和N16961）和代表性的El Tor变体（MQ1795和BAA-2163）在抗生素存在下表现出生长，并被认为对多粘菌素B具有抗性。将平板在37℃下孵育18小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55760/55760fig3.jpg" /> 图3:使用柠檬酸盐介质测定柠檬酸盐代谢。利用柠檬酸盐作为唯一碳源的能力通过分离物在最小柠檬酸盐培养基上生长的能力来确定。 WT经典菌株O395在最小柠檬酸盐培养基上不生长（阴性）。所有WT El Tor菌株（C6706和N16961）和代表性的El都显现了生长Tor变体（MQ1795和BAA-2163），其证明了利用柠檬酸盐作为唯一碳源（阳性）的能力。将板在37℃下孵育18小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55760/55760fig4.jpg" /> 图4:使用牛奶琼脂培养基测量HapR调节的蛋白水解酪蛋白水解。通过HapR调节的蛋白酶活性的酪蛋白水解通过围绕牛奶琼脂接种点的视觉区域来确定。含有非功能性HapR的菌株，如WT经典菌株O395和WT El Tor菌株N16961，在接种点（ hapR-阴性）周围没有产生清除区。 WT El Tor菌株C6706和代表性的El Tor变体（MQ1795和BAA-2163）含有功能性HapR，其可以被视为不同大小的清除区（ hapR阳性）。将板在37℃下孵育18小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55760/55760fig5.jpg" /> 图5:使用血琼脂培养基测量溶血活性。使用补充有绵羊血液的琼脂板测量溶血活性。 WT经典菌株O395不分泌裂解红细胞（γ-溶血）的酶。 WT El Tor菌株（N16961和C6706）和代表性的El Tor变体（MQ1795和BAA-2163）分泌溶血酶，其导致围绕接种点的半透明区域（β-溶血）。板在37℃下孵育°C 48小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55760/55760fig6.jpg" /> 图6:使用动力学琼脂平板确定运动性。运动性区域由盐TTC的视觉颜色变化表示，其在代谢时从透明变为红色，指示细菌移动的位置。 WT经典菌株O395（10mm）和WT El Tor菌株C6706（15mm）显示出最小的运动性，而El Tor菌株N16961（21mm）和代表性的El Tor变体（MQ1795（25mm）和BAA-2163（29mm） ）表现出相对于O395和C6706的超动力。将板在37℃下孵育18小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig6large.jpg" target="_blank">请点击</a>这里查看一个较大版本的这个数字。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55760/55760fig7.jpg" /> 图7:Voges-Proskauer测定。使用Voges-Proskauer测定法测定葡萄糖发酵产生的乙酰头孢菌素。 WT经典菌株O395和代表性的El Tor变体（MQ1795和BAA-2163）由于葡萄糖发酵（阴性）不产生乙偶姻。 WT El Tor菌株（N16961和C6706）产生副产物乙偶姻，可通过深红色变化（阳性）显现。将管在室温下孵育4小时。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55760/55760fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

该手稿除了进行一系列生物化学测定之外，还描述了适用于霍乱弧菌的维护技术，它们共同用于临床和环境V之间的快速可靠的分化。 霍乱弧菌生物型实验室设置。

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

laboratory-techniques-used-to-maintain-differentiate-biotypes-vibrio

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

10.8K 次观看.  Plymouth State University. 该手稿除了进行一系列生物化学测定之外，还描述了适用于霍乱弧菌的维护技术，它们共同用于临床和环境V之间的快速可靠的分化。 霍乱弧菌生物型实验室设置。 

视频: Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

I am Es.In MindUP, we study biology of human pathogens in non hosts environments. Many human pathogens after exiting their human host have to survive in the environment before infecting another host. So our goal is to better understand processes that control survival of human pathogens in the environment.And our goal is to then better understand transmission dynamics of infectious agents. So we are using Vibrio cholera, the cost of agent of diarrhea, disease, cholera as a model organism. Vibrio cholera is a facultative human pathogen and it has an aquatic lifecycle.Therefore, it provides us great opportunity to study the survival of human pathogen in a natural environment. There are many tools to study this organism. Its genome is completely sequenced and there are micro array representing every gene in the genome.And furthermore, there are many tools that we can manipulate the genome of this organism. My research program takes advantage of these tools and integrates them to study aquatic lifecycle of cholera. So in our experiments, we try to simulate the aquatic environments that organism lives in.And then we would like to know how does this organism so senses fluctuations in its environment with respect to nutrient load, temperature, salinity, or other biological stressors such as pages and tzo? So in aquatic environment, RIA cholera is found either in a free living form or in a surface attached form. And the surface attached forms are known as biofilms, which are simply composed of microorganisms and their extra polymeric substances, which can be exo polysaccharide and proteins and sometimes DNA.And this matrix acts as a glue and hold the organism together. And when grown in biofilm mode, these organisms are actually resistant to fluctuating parameters of their environments and they can be better protected from, for example, phage and proteasome probations. And these are factors that are controlling abundance of microorganisms in the environment.Some of the recent techno technological advance that helped us in our studies are the completion of the genome sequence of the organism and development of microray technology. Using these technologies, we can determine at the whole genome level how this organism responds to major fluctuations in its environment such as nutrient deprivation or changes in temperature. Or one of our major goal is to integrate our whole genome expression studies to a network analysis.So we would like to identify genetic regulatory network that controls response of to fluctuating environmental parameters. We would like to be able to model behavior of organism in the environment. And also we still do not know when in the environment with whom Vibra Cide interacts with.We do not know much about ecology of this organism, and we also do not know relevance of the genes and processes we identified to be important for survival of this organism in the laboratory to the real world. No are the genes that we identified express when Ria cholera is present in natural environment. We also would like to be able to identify and culture organisms that Vibrio Cora interacts with because in the environment, Vibrio Cora is a part of the complex microbial community.So I think understanding biology of Vibrio Cora in natural environment would be a, it's a challenging and it's critical for us to understand biology of the organism. Within the next five years, I hope that we would be able to better characterize op polysaccharide components of biofilms. For example, in Vibrio cholera, we still do not know structure of the op polysaccharide that is holding the cells together.We also do not know all the components of biofilm metrics. Eventually I think it be great to be able to quantify components of the biofilm metrics in natural biofilms of these organisms. I think another major challenge is to study, be OC in natural environment.And I hope that we would be able to take advantage of comparative genomic studies and combine whole genomic expression profiling comparative genomics to better understand evolution of IBR cholera as a pathogen.

霍乱弧菌：模式生物研究细菌的发病机制 - 访

科研

生物学

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

免疫与感染

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

用于维持和区分生物型的实验室技术

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霍乱弧菌：模式生物研究细菌的发病机制 - 访

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

TransFLP - A基因修改方法

培养和维护

对于检测到的敏感性和特异性显色琼脂培养基发展

诱导细胞分化和单细胞成像

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

对于电感受的制备快速协议