作者感谢马蒂亚斯鱿鱼设施的支持和迈克尔·哈德菲尔德和Kewalo海洋实验室在现场收集的援助，并为这个协议作出贡献的Ruby和麦克福尔-艺实验室的成员在这个手稿的意见，Gyllborg。在曼德尔实验室的工作是支持由NSF IOS-0843633。

1。细菌菌剂的制备<ol><li> 0天 前两天鱿鱼接种，板13琼脂上的LBS相关的细菌株。 </li><li>在25-28℃过夜孵育细菌。 </li><li> 第1天 接种一五，各殖民地3毫升磅玻璃文化管中等鲵株感染。准备作为备份的重复管。 </li><li> 第2天 （协调细菌步骤与鱿鱼步骤3.7-3.10 1.4-1.6） 1小时前接种，继代培养细菌1:80（37.5μL）3毫升的玻璃文化管定位，并成长为与曝气1小时。 </li><li>测量样品的OD 600到接种前。典型的测量0.3-0.6取决于应变。 </li><li>为目标接种3-5 x 10 3 CFU / ml的计算如下接种量:接种量（μl）= 1.25 /外径600（例如，外径600 = 0.5，计算接种量= 1.25/0.5 = 2.5微升）。这笔款项是直接添加到海水中含有步骤4.1鱿鱼。这种计算可能需要调整不同品系五鲵或更低或更高的水平比这里指定的接种。 </li></ol> 2。琼脂板的制备菌剂的枚举<ol><li>每次治疗，标签定位板（每治疗2）接种步骤4.1板样品。 </li><li>添加5％无菌板电镀珠。 </li></ol> 3。收集鱿鱼少年<ol><li>即时使用验光仪的海洋测量盐度和调整到35‰。 </li><li>过滤1 L即时使用的过滤装置和附加真空线或真空泵，生成过滤灭菌即时海洋（FSIO）的海洋。含氧水纷飞大力每个配药前。日可重复使用的E滤波器单元为2天。 </li><li>分装成每两（2）一次性抽样碗40-50毫升FSIO。一个标签为earlies和作为timelies之一。 </li><li>准备削减的移液器约1厘米最低的山脊，从上面的提示（见图3）获得少年鱿鱼的超过塑料转让移液器。这有利于更广阔的领域，鱿鱼，通过它可以通过后收集。放弃任何转移移液器，其中有一个粗略的外露表面。 </li><li>使用准备转让移液器，收集E。一夜之间，孵化和转移到的FSIO earlies碗scolopes。早幼龟蛋系统已经在超过1小时，和容易受污染的殖民五鲵在蛋系统。不要使用敏感的殖民实验earlies。 </li><li>检查蛋坦克每30-45分钟为新的幼体。确保所有幼体在每个C清除赫克。取出转移吸管，和存款到的FSIO timelies碗幼体。及时收集动物是殖民实验。 </li><li>当收集完成后（约45分钟后，黄昏），鱿鱼转移到主要实验室。经验殖民不间断3小时接种实验室的光照条件下的动物是有利的。 </li><li>每次治疗，准备一个碗与40毫升FSIO。加入鱿鱼检测碗（最高N = 40元一碗）。 </li><li>准备作为aposymbiotic（负）控制额外碗。 </li><li>每次治疗准备一个专用的移液管。 </li><li>安乐死在2％的乙醇额外鱿鱼。 </li></ol> 4。鱿鱼殖民<ol><li> 第2天-使用P10的Pipetman，分配到每碗鱿鱼（步骤3.8），每次治疗细菌计算等份（步骤1.5）。后，立即启动3小时定时器第一次接种。 </li><li>每次治疗，创建"旋涡"在专用的移液管一碗碗的边缘附近放置移液器，移液器上下反复搅拌约10秒的水和鱿鱼。彻底混合是至关重要的。 </li><li>从每碗板50μL到步骤2.2 LBS琼脂平板（技术复制，板治疗50％的的微升板）。孵育25-28°C过夜。 </li><li>准备洗碗（100毫升FSIO / EA），每次治疗。 </li><li>准备小瓶果蝇 （4毫升FSIO / EA）每个鱿鱼。 </li><li>整整3小时后，各自洗碗（所有治疗）转让的鱿鱼。这将停止接种。 </li><li>进行与FSIO转移自身的果蝇小瓶每一个人的鱿鱼。每次治疗使用指定的移液管。 </li><li> 果蝇瓶的托盘移动的鱿鱼设施的返回天/ N的飞行光周期的动物胚胎发育过程中经历。 </li><li> 第3天-准备果蝇瓶（4毫升FSIO / EA）为每个鱿鱼。 </li><li>在22-24小时后接种黄昏之前，每个鱿鱼转移到一个新的果蝇小瓶。每次治疗使用指定的移液管。 </li><li> 第4天 -准备标记1.5 ml离心管（1/squid）。 </li><li>在此之前黄昏在46-48小时后接种，测量和记录每个鱿鱼在果蝇小瓶（光度计一套6的整合与自动读取盖子封闭）的发光。 </li><li>作为阴性对照的背景发光，测量与FSIO的小瓶，不包含任何鱿鱼。 </li><li>体积约700μL步骤4.12至1.5毫升离心管中传输每个鱿鱼。移动纸板冷冻箱。一旦盖子被放置在包装盒上，不消除细菌驱逐光线索，它不WELL-理解。 </li><li>离心管冻结在-80°C过夜。 </li></ol> 5。殖民化水平的测定<ol><li>对于每个鱿鱼，准备两（2）离心管中，每475μLFSIO（或高压灭菌70％的即时海洋）。 </li><li>准备首先使用清理杵和删除总值的碎片和/或组织的Kimwipe杵。 </li><li>地方杵尖，含95％乙醇50毫升烧杯中。乙醇应增加约3厘米的高度。 </li><li>对于每一个杵，从烧杯中取出，擦拭与Kimwipe尖。 </li><li>沾杵成乙醇浴，删除和插入（尖）到离心管架，并允许完全干燥空气约15分钟。 </li><li>在离心管架鱿鱼解冻（最大N = 8）。 </li><li>如有必要，调节音量至700μL。 </li><li>从步骤5.5使用杵，扰乱动物组织，直到墨囊RUPtures（水会变成灰暗色）。 </li><li>删除杵，并确保所有的组织仍然在管。 </li><li>组织简要涡整整10秒（使用一个定时器）。 </li><li>允许组织要休息10分钟。该组织将解决留在溶液中的细菌和油墨。按照计算，细菌/油墨解决方案的研究[A]稀释（即700μLE. scolopes光在器官匀浆）。串行1:20稀释（[A]，[]）如下所述。 </li><li>为[A]稀释，添加25μL[一]步骤5.1中编写的离心管。旋涡。 </li><li>对于汇编[C]稀释，添加25μL[b]步骤5.1中编写的离心管。旋涡。 </li><li> 50板每到LBS的琼脂稀释液，2重复，每次治疗。 </li><li>在25-28℃过夜孵育板。 </li></ol> 6。数据分析<ol><li>计算CFU /光器官（LO），coun10-400殖民地ţ殖民地盘上每次治疗在目前，并使用相应的计算公式: 单位/ LO =（[一]板殖民地）×14; 单位/ LO =（[b]板的殖民地）×280; 单位/ LO =（论文集[C]板的殖民地）×5600。 </li><li>绘制单个数据点规模对数和中位数。 </li><li>数据往往不是正态分布，用不同的差异，离群可能含有生物有意义的信息。因此，非参数测试提供了一个有用的方法，以确定是否治疗差异显着。 </li><li>使用统计分析软件GraphPad棱镜。对于两种治疗方法，使用Wilcoxon秩和检验。对于大于两个疗程之间的比较，使用适当的测试后，Kruskal-Wallis检验。 </li></ol> 7。代表结果从样品殖民检测结果如图4所示。两株<em&gt;五。鲵，表现出不同的发光相对水平是每个接种到6鱿鱼，以及作为aposymbiotic（uncolonized）控制六个鱿鱼。的大肠杆菌 ES114 14 scolopes共生，和光明Sepiola罗布斯塔共生的，SR5的15,16。类似的接种水平（图4A）3小时内导致100％的殖民。在48小时，发光水平（图4B）和CFU计数（图4C）被确定为殖民的应变能力的评估。允许每个过程中的共生菌的亮度测定的具体发光（图4D每细菌）的测定。 <img alt="图1" src="/files/ftp_upload/3758/3758fig1.jpg" /> 图1。殖民化过程的流程图。细菌和鱿鱼收获分开，然后在指定的接种混合。鱿鱼洗净，然后转移到新的水在3小时，24小时和4<html> 8小时后接种。在48小时发光测量和动物被冻结，其作用是表面消毒的动物。光器官定植细菌仍然可行-80°Ç通过一个解冻（没有额外的冻融）。 <img alt="图2" src="/files/ftp_upload/3758/3758fig2.jpg" /> 图2。流程图，说明细菌的同质化和稀释电镀。定植细菌的枚举序列的20倍稀释液提供适当的动态范围。 <img alt="图3" src="/files/ftp_upload/3758/3758fig3.jpg" /> 图3。适当窄轴锥度移液器转移到一个狭窄的孔（一），会损害青少年的鱿鱼。最窄的部分用剪刀或刀片切断预处理产生一个合适的工具（B）为转移少年鱿鱼。 s/ftp_upload/3758/3758fig4.jpg"/&gt; 图4为殖民检测样本数据。 （一）细菌水平在接种碗。 （二）个别鱿鱼发光。 （三）个别鱿鱼的菌落计数。 （四）具体的个别鱿鱼发光。 APO，Aposymbiotic的（uncolonized阴性对照）。

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作者没有透露。

描述的殖民法，允许在受控的实验室环境中的一种自然的共生过程的分析。因此，它可以用来评估由不同的自然株，突变株，并根据不同的化学制度的殖民化。通常用来描述实验上的变化，以评估不同方面的共生。可以测量殖民动力学检查在第一个24小时，它可以自动检测巧合探测器已被删除，在闪烁计数器的发光。此外，一个应变，相对于其他相对定植能力，可以通过有竞争力的殖民实验测量?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>玻璃培养管，直径16毫米</td><td>厂商VWR </td><td> 47729-580 </td><td></td></tr><tr><td>上限为玻璃培养管</td><td>费舍尔</td><td> NC9807998 </td><td></td></tr><tr><td>可见分光光度计测定OD值600 </td><td> biowave </td><td> CO8000 </td><td>任何能够测量外径600分光光度计的工作。本机可直接测量液体的OD 600在玻璃培养管。一些接种计算的调整可能是必要的，取决于所使用的仪器。 </td></tr><tr><td> GloMax 20/20单管光度计</td><td> Promega公司</td><td> E5311 </td><td>相当于Turner公司20/20n光度计。包括离心管架。 </td></tr><tr><td> GloMax 20/20晚间标准</td><td> Promega公司</td><td> E5341 </td><td>用于光度计校正。 </td></tr><tr><td>折射仪，手持式</td><td>福斯特和史密斯游泳</td><td> CD-14035 </td><td>校准之前，每次使用去离子水。使用后每用去离子水冲洗，以防止盐积聚。 </td></tr><tr><td>即时海洋（人工海水浓缩液） </td><td>福斯特和史密斯游泳</td><td> CD-16881 </td><td>准备在35‰，在去离子水，使用的验光仪，通过SFCA 0.2微米的过滤器，然后过滤。 </td></tr><tr><td>过滤装置</td><td> NALGENE </td><td> 158-0020 </td><td>表面活性剂醋酸纤维素（SFCA）膜，0.2微米。我们已经观察到一些含表面活性剂的聚醚砜过滤器变量的结果。 </td></tr><tr><td>转移移液器</td><td>费舍尔</td><td> 13-711 -上午9时</td><td>用剪刀或刀片，切干净以上第一山脊增加枪头直径和避免挤压鱿鱼幼体的一角。 </td></tr><tr><td>一次性样品碗（塑料酒杯） </td><td>彗星</td><td> T9S（9盎司）。 </td><td>接种碗，上直径3¼"，直径2¼"，高度为3"。碗创建一个同质的环境，因为他们没有底部的轮辋，其中鱿鱼可以得到被困在低氧气小生。大小进行了优化40毫升接种。可用在webstaurantstore.com，619PI9。 </td></tr><tr><td> 果蝇样品瓶</td><td>厂商VWR </td><td> 89092-720 </td><td>瓶直径相匹配的光电倍增管上的光度计开幕。 </td></tr><tr><td> 1.5毫升离心管</td><td> ISC的Bioexpress </td><td>的C-3217-1CS </td><td>管必须符合杵的形状。 </td></tr><tr><td>乙醇，200证明</td><td>费舍尔</td><td>端口BP2818-100 </td><td></td></tr><tr><td>杵</td><td>金布尔大通/ Kontes </td><td> 749521-1500 </td><td></td></tr><tr><td>电镀珠，直径为5毫米</td><td>金布尔大通</td><td> 13500 5 </td><td>每管和高压灭菌器，准备5。 </td></tr></tbody></table>

colonization of euprymna scolopes squid by vibrio fischeri

与特定的细菌被发现在动物组织1-5。这种细菌主机协会（共生）可能是有害的（致病性），有没有健身的后果（共生），或有利于（互惠）。而备受人们关注已给病原相互作用，知之甚少的进程决定的有利/共生细菌的环境重现收购。轻器官之间的海洋革兰氏阴性菌共生五鲵和夏威夷短尾鱿鱼，E. scolopes，代表着一个非常具体的互动，其中一台主机（E. scolopes）建立贯穿其一生当然6,7只有一个菌种（ 五鲵 ）的共生关系。由五，生产发光鲵在这种互动提供了一个五反掠夺利益在8,9夜间活动scolopes，而营养丰富的宿主组织提供V。鲵与受保护的利基10。在每个主机上一代，这一关系的概括，从而较可预测的过程，可以在共生发展的各个阶段的详细评估。少年鱿鱼孵化aposymbiotically（uncolonized），并在实验室中，如果前30-60分钟内收集和转移到共生的自由水，不能被殖民除实验接种6。因此，这种互动提供了一个有用的模型系统，在其中进行评估，导致从11,12环境的共生微生物的具体收购的各个步骤。 在这里，我们描述了一种方法来评估发生刚出时aposymbiotic 大肠杆菌殖民化的程度scolopes暴露（人造）海水中含有五。鲵。这个简单的实验说明接种，自然感染和恢复E.新生的光器官的细菌共生scolopes。护理提供一个一致的环境共生的发展过程中的动物，尤其是关于水质和轻线索。特征描述的共生人口的方法包括:（1）的细菌派生发光的测量，（2）直接菌落计数恢复共生。

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

方法概括的过程，其中夏威夷短尾鱿鱼，<em&gt; Euprymna scolopes</em&gt;和其细菌的共生体，<em&gt;弧菌鲵</em&gt;，分别提出，然后介绍了允许由细菌鱿鱼轻机关的具体殖民。定植细菌衍生的发光性能和直接菌落计数检测。

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

colonization-of-euprymna-scolopes-squid-by-vibrio-fischeri

Research

JoVE Journal

Immunology and Infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri

19.8K 次观看.  Northwestern University. 方法概括的过程，其中夏威夷短尾鱿鱼， Euprymna scolopes和其细菌的共生体，弧菌鲵，分别提出，然后介绍了允许由细菌鱿鱼轻机关的具体殖民。定植细菌衍生的发光性能和直接菌落计数检测。

视频: Colonization of Euprymna scolopes Squid by Vibrio fischeri

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

科研

免疫与感染

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

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 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

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

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

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

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

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 ...

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 ...