وأيد هذا العمل جمعية ماكس بلانك.

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هذه الورقة تقارير طريقة التصوير المجهري للخلايا بارايموليتيكوس V. بارايموليتيكوس ، تليها التحليلات المصب تهدف إلى حل وتحديد توطين تحت الخلوية وغيرها من الميزات من البروتينات فلورزنتلي المسمى درس. على الرغم من وجود العديد من الأدوات لمعالجة الصور المجهرية وتحل...

البكتيريا باستمرار تجربة التغييرات على البيئة الخارجية، وقد وضعت العديد من التقنيات لتغيير وتكييف سلوكهم وفقا لذلك. واحدة من هذه الآلية تنطوي على التمايز إلى أنواع الخلايا المتميزة التي تكمل بشكل أفضل البيئة المتغيرة. التمايز غالبا ما ينطوي على تغييرات كبيرة في تنظيم دورة الخلية، مورفولوجيا الخلية، والتنظيم الزماني الزماني للخلايا. أحد الكائنات الحية التي يمكن أن تخضع للتمايز هو فيبرايو بارايموليتيكوس . V. بارايموليتيكوس ينتمي إلى فيبريوناسي ، الذي هو عائلة من بروتيوباكتيريا التي تسكن عادة المياه العذبة أو المالحة. يتم توزيع فيبريوناسي على نطاق واسع في البيئة وتشمل العديد من الأنواع التي تسبب التهابات المسالك المعوية في البشر، بما في ذلك أيضا ضمة الكوليرا. باراهايموليتيكوس هو كائن حيوي و قادر على التفريق في نوعين مختلفين من الخلايا كرد فعل لاستيعاب التغيرات فيالوسط الخارجي. في البيئات المائية، فإنه موجود كخلايا السباح القصيرة على شكل قضيب مع ساق القطبية واحدة المتمركزة في القطب الخلية القديمة. عند الاتصال السطح، يتم تشغيل التمايز في خلية سرب. سوارمر تمايز الخلايا ينطوي على اثنين من التغييرات الرئيسية: سربار خلية التشكل من خلال تثبيط انقسام الخلايا، وتحريض نظام سوط الثاني. ويؤدي ذلك إلى تكوين خلية أسماك ذات شكل قضيب بيريتريشوس وممدود للغاية، والتي يمكن أن تستمر إما في نمط حياة القطيع، حيث تؤدي أحداث الانقسام إلى خلايا أسلاف أسلاف، أو تفرق في الخلف إلى خلايا السباح. وقد تم الإبلاغ عن عدة عوامل لتحفيز أو التأثير على التمايز أسوأ. ويبدو أن الحافز الأساسي يقودها ميكانوسنسينغ حيث V. بارايموليتيكوس يستخدم العلم القطبي كمستشعر اللمس الذي يكشف عن تثبيط دوران على اتصال السطح، ولكن هناك عدة عوامل أخرى تشارك كماحسنا 1 . في المختبر، دوران الساق القطبية يمكن أن يتم تثبيطها بشكل مصطنع بإضافة فيناميل، الذي يحجب دوران الصفيحة التي تحركها الصوديوم، مما يؤدي إلى تمايز الأسوار 2 . وعلاوة على ذلك، في دراسة سابقة، فيبريو ألجينوليتيكوس كان يحرض على سرب على وسائل الاعلام الصلبة عندما تم نشر الخلايا على وسط النمو في طبق بتري مختومة بشريط بلاستيكي واضح. ومنعت ورقة المرشح المشبعة بالقلويات من الاحتشاد في ظل هذه الظروف، مما يوحي بأن واحدا أو أكثر من الأحماض المتطايرة قد يكون ضالعا في تحريض الاحتكاك. وبالتالي، فإن ختم طبق بتري مع الشريط البلاستيك المرجح للسماح لتراكم الأحماض المتطايرة، التي شكلت من قبل منتجات ثانوية من التمثيل الغذائي الخلوي، داخل مساحة الرأس من لوحة. وقد تحقق نفس التأثير عندما أضيف H2 O 2 إلى وسط النمو في أطباق بيتري غير مختومة من أجل إنتاج الأحماض المتطايرة بشكل مصطنع عن طريق هدروليسينغ المكونات الإعلامية <supكلاس = &quot;كريف&quot;&gt; 3 ، 4 ، 5 . ومع ذلك، لا تزال هوية هذه الأحماض المتطايرة غير معروفة. وعلاوة على ذلك، فقد تبين أن توافر الفائض من الكالسيوم 6 والحد من الحديد 7 على حد سواء تعزيز التمايز أسوأ وانتشار على الأسطح الصلبة. يمكن تجويع الخلايا للحديد بإضافة مركب 2،2&#39;-بيبيريديل إلى وسط النمو، والذي ثبت أنه يؤثر على تمايز الأسوار 8 . يتم الآن تنفيذ العوامل المعروفة لتنظيم التمايز في تصميم بروتوكول الذي يحفز على نحو متكاثر V. التمايز بارايموليتيكوس في الخلايا الأبقار وانتشارها على السطوح أجار الصلبة. V. بارايموليتيكوس التمايز ينطوي على تغييرات كبيرة في تنظيم انقسام الخلايا، مورفولوجيا الخلوية، وتحديد المواقع من الآلات الجزيئية مثل فلاجيلأ و الكيميائي الأجهزة - العمليات التي تتطلب كل توطين محددة من البروتينات وفقا لدورة الخلية. وبالتالي، فإن القدرة على دراسة توطين الخلايا من هذه البروتينات أمر ضروري لفهم العمليات الخلوية المذكورة أعلاه. من أجل إجراء مثل هذه الدراسات مضان المجهري على خلايا واحدة مطلوب. هذا يمكن أن يكون تحديا بشكل خاص عندما خلايا التصوير الموجودة داخل السكان البكتيرية الكثيفة، كما هو الحال بالنسبة للخلايا أسوأ. كانت هناك محاولات للحث على التمايز أسوأ في وسائل الإعلام السائل، والتي يمكن أن تولد خلايا واحدة للدراسات المجهري. وعلى الرغم من أن على مستوى النسخي هذه الخلايا قد تسببت جزئيا برنامج تمايز أسراب، فإنها لا تخضع لنفس التغييرات المورفولوجية متميزة كما خلايا سربار متباينة تماما نمت على المتوسطة الصلبة 8 . تقدم هذه الورقة بروتوكول قوي وقابلة للتكرار من طريقة للحث على سربار مليرة لبنانية التمايز على سطح أجار. بروتوكول ينتج السكان من الخلايا التي يسهل الوصول إليها بسهولة أكثر سربار متاحة بسهولة للمجهر الخلية واحدة والتحليل اللاحق. وعلاوة على ذلك، بروتوكول تمكن دراسات التعريب من البروتينات فلورزنتلي المسمى في كل من السباح وأنواع الخلايا أسوأ (أقسام 1، 2، 3، 4). بالإضافة إلى ذلك، يصف البروتوكول سير العمل اللاحقة على كيفية معالجة وتحليل البيانات التي تم إنشاؤها من التجارب المجهري مضان، والذي يسمح للتحليل الديموغرافي للمجتمعات البكتيرية (القسم 5).

<table><tbody><tr><td>&lt;strong&gt;Media components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LB medium</td><td>Roth</td><td>X968.3</td><td></td></tr><tr><td>Difco Agar, granulated</td><td>BD</td><td>214510</td><td>For preparation of LB agar with LB medium</td></tr><tr><td>Difco Heart Infusion Agar</td><td>BD</td><td>244400</td><td>For preparation of HI agar swarming plates</td></tr><tr><td>Agarose NEEO, ultra quality</td><td>Roth</td><td>2267.3</td><td>For preparation of microscopy agarose pads</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Additives&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L-arabinose</td><td>Roth</td><td>5118.3</td><td>Used to induce pBAD derivatives</td></tr><tr><td>2,2`-Bipyridyl</td><td>Sigma Aldrich</td><td>D216305-25G</td><td>For preparation of HI agar swarming plates</td></tr><tr><td>Calcium chloride dihydrate</td><td>Roth</td><td>5239.1</td><td>For preparation of HI agar swarming plates</td></tr><tr><td>Ampicillin sodium salt</td><td>Roth</td><td>K029.3</td><td></td></tr><tr><td>Chloramphenicol</td><td>Roth</td><td>3886.3</td><td></td></tr><tr><td>PBS buffer</td><td>-</td><td>-</td><td>Standard recipe for Phosphate-buffered saline</td></tr><tr><td>D(+) Sucrose</td><td>Roth</td><td>4621.1</td><td>For preparation of electrocompetent cells</td></tr><tr><td>Glycerol</td><td>Roth</td><td>3783.5</td><td>For preparation of electrocompetent cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Hardware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Petri dish 150 mm x 20 mm with cams</td><td>Sarstedt</td><td>82.1184.500</td><td>For preparation of HI agar swarming plates</td></tr><tr><td>kelvitron t (B 6420)</td><td>Heraeus</td><td>-</td><td>Used for drying HI agar swarming plates</td></tr><tr><td>Gene Pulser Xcell Microbial System</td><td>BioRad</td><td>1652662</td><td>Used for elctroporation of plasmid DNA</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MetaMorph Offline (version 7.7.5.0)</td><td>Molecular Devices</td><td>-</td><td>Used for microscopy image analysis</td></tr><tr><td>Excel</td><td>Microsoft</td><td>-</td><td>Used for microscopy data analsis</td></tr></tbody></table>

induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells

القدرة على دراسة توطين الخلايا من البروتينات ضروري لفهم العديد من العمليات الخلوية. في المقابل، وهذا يتطلب القدرة على الحصول على خلايا واحدة للمجهر مضان، والتي يمكن أن تكون صعبة بشكل خاص عندما خلايا التصوير التي توجد داخل المجتمعات البكتيرية. على سبيل المثال، المسببة للأمراض البشرية فيبريو باراهايموليتيكوس موجود كخلايا السباح القصيرة على شكل قضيب في الظروف السائلة التي على سطح الاتصال التفريق في سوبوبولاتيون من خلايا أسد ممدود للغاية المتخصصة للنمو على الأسطح الصلبة. تقدم هذه الورقة طريقة لأداء تحليل خلية واحدة مضان المجهري من V. بارايموليتيكوس في دولتي التفاضلية. هذا البروتوكول استنساخا جدا يستحث التمايز من V. بارايموليتيكوس في الخلية الدافع حياة دورة ويسهل انتشارها على الأسطح الصلبة. تنتج هذه الطريقة مشاعل الخلايا المتمايزة الأيسر تمتد من tوقال انه حافة مستعمرة سرب. ومن الجدير بالذكر، في طرف جدا من سرب مشاعل، توجد خلايا أسوأ في طبقة واحدة من الخلايا، والذي يسمح لنقلها السهل إلى شريحة المجهر والتصوير المجهري مضان اللاحقة من خلايا واحدة. بالإضافة إلى ذلك، يتم عرض سير العمل لتحليل الصور للتمثيل الديمغرافي للمجتمعات البكتيرية. وكدليل على المبدأ، يتم وصف تحليل توطين الخلايا من الكيميائي يشير صفائف في السباح والخلايا الداعمة من V. بارايموليتيكوس.

تحريض التمايز وتوليد المستعمرات الشكل 1 يوفر مخططا من الخطوات الهامة التي ينطوي عليها إنتاج مستعمرات سرب من V. بارايموليتيكوس ( الشكل 1A-C ). وقد رصدت ثقافة السباح على سر...

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

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

هذا البروتوكول يتيح المجهر خلية واحدة من فيبريو باربرايموليتيكوس السباح متميزة متميزة وخلايا أسوأ. وتنتج هذه الطريقة مجموعة من الخلايا الداعمة المتاحة بسهولة لتحليل خلية واحدة ويغطي إعداد الثقافات الخلية، تحريض التمايز سوبيرمر، وإعداد العينات، وتحليل الصور.

تحريض التمايز الخلوي والتصوير خلية واحدة من<em&gt; فيبرايو بارايموليتيكوس</em&gt; السباح و سوارمر الخلايا

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

induction-cellular-differentiation-single-cell-imaging-vibrio

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

9.3K Views.  Max Planck Institute for Terrestrial Microbiology.  هذا البروتوكول يتيح المجهر خلية واحدة من فيبريو باربرايموليتيكوس السباح متميزة متميزة وخلايا أسوأ. وتنتج هذه الطريقة مجموعة من الخلايا الداعمة المتاحة بسهولة لتحليل خلية واحدة ويغطي إعداد الثقافات الخلية، تحريض التمايز سوبيرم...

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

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

مراقبة المكانية الفصل في استعمار سطح الميكروبية السكان

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetics

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

قياس حركة احتشاد السطح البكتيري على ألواح التدرج المحفزة

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

Biochemistry

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

شريحة موائع دقيقة بسيطة للنمو على المدى الطويل وتصوير Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioengineering

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

التصور الحركي لتفاعلات البكتيريا بين الخلايا المفردة

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

التحليل الآلي للأنماط الظاهرية داخل الخلايا للسالمونيلا باستخدام ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

التحضير والتصوير، والكمي لالسطحية الجرثومي على الحركة فحوصات

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Biology

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

التمييز وتوصيف السكان غير المتجانسة باستخدام تقنيات التصوير الكمي

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

تحليل موائع جزيئية خلية واحدة نجحت عصية

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

التصوير الحي للخلايا من دورة حياة البكتيريا المفترس Bdellovibrio bacteriovorus باستخدام المجهر الفلوري الوقت الفاصل

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

التحقيق في حركية Flagella يحركها في الإشريكية القولونية من خلال تطبيق ثلاث تقنيات راسخة في سلسلة

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

تحريض التمايز الخلوي والتصوير خلية واحدة من

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