Name: visualization of biofilm formation in candida albicans using an automated microfluidic device
Uploaded: 2017-12-14T15:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

ونشكر جميع أعضاء المختبر Nobile لإجراء مناقشات مفيدة في فحوصات بيوفيلم. وأيد هذا الدراسة المعاهد الوطنية للصحة (المعاهد الوطنية للصحة) منحة R21 AI125801 (C.J.N.). وأيد D.L.R. على زمالة دكتوراه من جامعة "معهد كاليفورنيا" للمكسيك والولايات المتحدة (UC-مكسيوس) y Consejo ناسيونال دي العلوم تكنولوجيا (المجلس الوطني).

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E., Marchillo, K., Spiegel, C. A., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+in+vivo+Candida+albicans+biofilm+denture+model.">Development and validation of an in vivo Candida albicans biofilm denture model.</a> Infect Immun. 78 (9), 3650-3659 (2010).</li><li>Nett, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+indwelling+urinary+catheter+model+of+Candida+albicans+biofilm+infection.">Rat indwelling urinary catheter model of Candida albicans biofilm infection.</a> Infect Immun. 82 (12), 4931-4940 (2014).</li><li>Krom, B. P., Willems, H. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45 (9), 2475-2479 (2001).</li><li>Nett, J. E., Cain, M. T., Crawford, K., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+Candida+biofilm+microtiter+plate+model+for+measurement+of+antifungal+susceptibility+by+tetrazolium+salt+assay.">Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay.</a> J Clin Microbiol. 49 (4), 1426-1433 (2011).</li><li>Krom, B. P., Cohen, J. B., McElhaney Feser, G. E., Cihlar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+candidal+biofilm+microtiter+assay.">Optimized candidal biofilm microtiter assay.</a> J Microbiol Methods. 68 (2), 421-423 (2007).</li><li>Lohse, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+Optimizations+of+Candida+albicans+In+Vitro+Biofilm+Assays.">Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays.</a> Antimicrob Agents Chemother. 61 (5), (2017).</li><li>Winter, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Identification+of+Biofilm-Specific+Proteolysis+in+Candida+albicans.">Global Identification of Biofilm-Specific Proteolysis in Candida albicans.</a> mBio. 7 (5), (2016).</li><li>Nobile, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recently+evolved+transcriptional+network+controls+biofilm+development+in+Candida+albicans.">A recently evolved transcriptional network controls biofilm development in Candida albicans.</a> Cell. 148 (1-2), 126-138 (2012).</li><li>Fox, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+regulatory+network+temporally+controls+Candida+albicans+biofilm+formation.">An expanded regulatory network temporally controls Candida albicans biofilm formation.</a> Mol Microbiol. 96 (6), 1226-1239 (2015).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Curr Biol. 15 (12), 1150-1155 (2005).</li><li>Baker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the bench: A laboratory navigator. 27, (2005).</li></ol>

كلاريسا J. Nobile مؤسس بيوسينيسيس، شركة، وهي شركة تطوير مثبطات والتشخيص للأغشية الحيوية المبيضة .

والرزن بيوفيلم موائع جزيئية للتخصيص الموضحة هنا يسمح لتصور تشكيل بيوفيلم في الوقت الحقيقي على مستوى خلية واحدة عندما تتعرض إلى معدل ثابت الاندفاق الصفحي ودرجة حرارة ثابتة. ويوفر وسيلة قوية لدراسة تطوير الأغشية الحيوية في السلالات البرية من نوع ومتحولة، ولوحظت آثار المعالجات عامل مضادات...

المبيضات البيض عضو commensal الحجمية البشرية، إلا وهو أيضا الممرضات انتهازية، القادرة على التسبب بالأمراض الفطرية السطحية وحادة1،2. سمة فوعة رئيسية من المبيضة هو قدرته على نموذج مرن والأغشية الحيوية مقاومة للعقاقير، مجتمعات خلايا انضمت إلى سطح والمغلقة في مواد1،مصفوفة خارج الخلية3. الأغشية الحيوية المبيضة عالية منظم، الذي يحتوي على عدة طبقات من أنواع متعددة من الخلايا (جولة الناشئين الخميرة على شكل الخلايا والخلايا بسيودوهيفال البيضاوي وخلايا أصله أنبوبي)4. المبيضة بيوفيلم التنمية يبدأ مع التمسك بخلايا الخميرة جولة-نموذج سطح (البذر في بيوفيلم)، تليها انتشار هذه الخلايا على السطح، وثم هيكل نضوج بيوفيلم غير ناضجة في بيوفيلم تشكيل تماما محاط ب المواد المصفوفة خارج الخلية4. بيوفيلم ناضجة الغالب تتألف من خلايا أصله ممدود التي تشكل شبكات كثيفة ومترابطة، وتوفير الاستقرار المعمارية إلى بيوفيلم4. طوال دورة الحياة بيوفيلم، جولة الناشئين الخميرة الخلايا تفريق من بيوفيلم ناضجة، ويجوز السفر إلى مناطق أخرى من الجسم لتسبب التهابات نشرها أو البذور الأغشية الحيوية الجديدة في،من4مواقع أخرى5. يمكن أن تشكل المبيضة الأغشية الحيوية على السطوح الحيوية، مثل الأسطح المخاطية وفي جميع أنحاء أنسجة المضيف، وعلى الأسطح غير الأحيائية، مثل القسطرة ومنظم نبضات القلب، وأطقم الأسنان والمفاصل الاصطناعية. بسبب خصائص المعاندة للأغشية الحيوية، أنها صعبة للغاية للقضاء على، وفي كثير من الحالات أن استراتيجية العلاج الفعال الوحيد إزالة الأجهزة المصابة4. وبالتالي من الأهمية بمكان للتحقيق في تشكيل بيوفيلم تحت ظروف مماثلة لتلك التي لوحظت في إعدادات السريرية.
وهناك عدة حاسمة في فيفو النماذج الحيوانية المستخدمة لدراسة المبيضة بيوفيلم تشكيل6،،من78؛ بيد أن هذه الدراسات يمكن أن تكون مكلفة وتستغرق وقتاً طويلاً، وتقتصر على عدد من سلالات ومضادات الجراثيم التي يمكن اختبارها في وقت معين. في المختبر بيوفيلم فحوصات، من ناحية أخرى، تسمح للتقييم السريع والفائق للمركبات المضادة للفطور وسلالات متحولة، وهي أكثر فعالية من حيث التكلفة والأخلاقية من فحوصات بيوفيلم التي تقوم بها في الحيوان نماذج9، 10،،من1112،،من1314. هنا يصف لنا في المختبر مقايسة التي وضعت والأمثل لمراقبة تشكيل بيوفيلم وقتيا تحت الاندفاق الصفحي استخدام موائع جزيئية لتخصيص جهاز14،15. يسمح الفحص للتصور من كل مرحلة من مراحل تكوين بيوفيلم، بما في ذلك الخطوة الأولى التقيد وتكاثر الخلايا، ونضوج بيوفيلم، وتشتت الخلية. المقايسة مفيد أيضا لتصور التغييرات مورفولوجيا الخلية في جميع أنحاء تطوير بيوفيلم.
لوحات ميكروتيتير، والتي تستخدم عادة في المختبر فحوصات بيوفيلم، بينما إنتاجية عالية، لا تسمح لظروف التدفق التي تسيطر عليها. تسمح أنظمة الخلية الاندفاق الصفحي التقليدية للتقييم المستمر لتشكيل بيوفيلم في ظروف التدفق التي تسيطر عليها، ولكن هذه غالباً ما تستغرق وقتاً طويلاً لإعداد وتميل إلى التحكم الديناميكي وإنتاجية محدودة. الجهاز موائع جزيئية المستخدمة هنا يتغلب على هذه القيود من خلال الجمع بين لوحات الإنتاجية العالية (التي تحتوي على آبار 48) مع دائرة الاندفاق الصفحي مدمج واستنساخه عاليا وتنوعاً، وقابلة للتخصيص.
وهنا، يمكننا وصف بروتوكول لاستخدام جهاز موائع جزيئية الآلي المتاحة تجارياً لتقييم تكوين بيوفيلم البرية من نوع المبيضة سلالة، آثار عامل مضاد فطري معروف على تطوير بيوفيلم، وبيوفيلم تشكيل في سلالات متحولة اثنين (bcr1Δ/Δ و efg1 Δ/Δ) التي تم الإبلاغ عنها سابقا أن يكون بيوفيلم العيوب في المختبر و في فيفو16،،من1718. يمكن استخدامها في وصف بروتوكول اختبار فعالية مضادات الجراثيم في تثبيط تكوين بيوفيلم في جميع أنحاء تطوير بيوفيلم، وتحديد الجينات المطلوبة للتنمية بيوفيلم العادي بفحص مكتبات متحولة.

<table><tbody><tr><td>BioFlux 1000z</td><td>Fluxion</td><td></td><td>Automated microfluidic device for live cell analysis</td></tr><tr><td>48-well plate 0-20 dyne</td><td>Fluxion</td><td>910-0047</td><td>Microfluidic plate</td></tr><tr><td>Montage Software</td><td>Fluxion</td><td>Version 7.8.4.0</td><td>Visualization analysis software</td></tr><tr><td>ImageJ Software</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td><td></td></tr><tr><td>Yeast Extract</td><td>Criterion</td><td>C7341</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD Biosciences</td><td>211677</td><td></td></tr><tr><td>Dextrose (D-Glucose)</td><td>Fisher Scientific</td><td>D163</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R6504</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>Nutrient Broth</td><td>Criterion</td><td>C6471</td><td></td></tr><tr><td>Difco D-Mannitol</td><td>BD Biosciences</td><td>217020</td><td></td></tr><tr><td>Agar</td><td>Criterion</td><td>C5001</td><td></td></tr><tr><td>Amphotericin B</td><td>Corning</td><td>30-003-CF</td><td></td></tr><tr><td>Sterile Inoculating Loops</td><td>VWR</td><td>30002-094</td><td></td></tr><tr><td>Petri Dishes with Clear Lid</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Disposable Cuvettes</td><td>Fisher Scientific</td><td>14-955-127</td><td></td></tr><tr><td>Lens Paper</td><td>VWR</td><td>52846-001</td><td></td></tr><tr><td>Microplate and Cuvette Spectrophotometer</td><td>BioTek</td><td>EPOCH2TC</td><td></td></tr><tr><td>Shaking Incubator</td><td>Eppendorf</td><td>M12820004</td><td></td></tr></tbody></table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

المبيضات البيض هي الأكثر شيوعاً الممرض الفطرية للبشر، مما تسبب في حوالي 15% حالات الانتان المكتسبة في المستشفى. هو سمة فوعة رئيسية من المبيضة لها القدرة على شكل الأغشية الحيوية، والمجتمعات تنظيماً للخلايا التي تعلق على السطوح الحيوية وغير الحيوية. يمكن أن تشكل الأغشية الحيوية المبيضة على أنسجة المضيف، مثل الطبقات المخاطية، والأجهزة الطبية، مثل القسطرة ومنظم نبضات القلب، وأطقم الأسنان والأطراف الصناعية المشتركة. الأغشية الحيوية تطرح تحديات سريرية هامة لأنها شديدة المقاومة للاضطرابات الفيزيائية والكيميائية، ويمكن أن تعمل كخزانات للبذور نشر العدوى. وقد استخدمت مختلف في المختبر فحوصات لدراسة تكوين بيوفيلم المبيضة ، مثل microtiter لوحة فحوصات وقياسات الوزن الجاف وفحوصات سلامة الخلية ومجهرية ليزر المسح [كنفوكل]. كل من هذه الاختبارات فحوصات نقطة نهاية واحدة، حيث يتم تقييم تشكيل بيوفيلم في نقطة زمنية محددة. وهنا يصف لنا بروتوكولا لدراسة تشكيل بيوفيلم في الوقت الحقيقي باستخدام جهاز موائع جزيئية الآلي تحت ظروف التدفق الصفحي. يسمح هذا الأسلوب للمراقبة لتشكيل بيوفيلم بيوفيلم يتطور مع مرور الوقت، استخدام الشروط القابلة للتخصيص التي تحاكي تلك المضيفة، مثل تلك التي ووجهت في القسطرة والأوعية الدموية. يمكن استخدام هذا البروتوكول لتقييم عيوب بيوفيلم طفرات وراثية وكذلك الآثار المثبطة لمضادات الجراثيم في التنمية بيوفيلم في الوقت الحقيقي.

أجرينا المقايسة بيوفيلم موائع جزيئية الموصوفة هنا باستخدام نوع البرية سلالة المبيضة ظروف اثنين من وسائل الإعلام (media ربمي-1640 والعنكبوت)، وسلالة البرية من نوع حضور العقاقير المضادة للفطور المعروفة الامفوتريسين ب (16 ميكروغرام/مل) في ربمي، وسبق أن أبلغت سلالات متحولة ?...

Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

التصور "تشكيل بيوفيلم" في المبيضات البيض باستخدام جهاز موائع جزيئية الآلي

ويصف هذا البروتوكول استخدام جهاز موائع جزيئية الآلي للتخصيص لتصور تشكيل بيوفيلم في المبيضات البيض تحت ظروف فسيولوجية المضيف.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

visualization-biofilm-formation-candida-albicans-using-an-automated

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JoVE Journal

Immunology and Infection

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

10.5K Views.  University of California, Merced. ويصف هذا البروتوكول استخدام جهاز موائع جزيئية الآلي للتخصيص لتصور تشكيل بيوفيلم في المبيضات البيض تحت ظروف فسيولوجية المضيف.

Video: Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

96 ألف لوحة حسنا Microtiter القائم على أسلوب تشكيل لمراقبة واختبار قابلية مضاد المبيضات البيض الأغشية الحيوية

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

المبيضات البيض بيوفيلم رقاقة (كاليفورنيا BChip) لالفرز الفائق الإنتاجية مضاد للمخدرات

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

بروتوكول لتشكيل بيوفيلم الملون في جهاز ميكروفلويديك مع مايكرو الأركان

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

استخدام إنتاجية عالية<em&gt; في المختبر</em&gt; نظام ميكروفلويديك لتطوير الأغشية الحيوية متعدد الأنواع عن طريق الفم

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

المبيضات البيض بيوفيلم التنمية على الهيئات الأجنبية طبيا ذات الصلة في الماوس تحت الجلد نموذج تلاه ضوء بارد التصوير

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Medicine

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

التصوير في الوقت الحقيقي والتحديد الكمي للتنمية بيوفيلم الفطرية باستخدام نظام تدفق دائرية ذات مرحلتين

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

توضيح والتصوير المبيضات ألبيكانس بيوفيم

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

مقايسة اختزال قابلة للذوبان تعتمد على التترازوليوم لتقييم تأثير الأجسام المضادة على الأغشية الحيوية المبيضات الاستوائية

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app.jove.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

المبيضات البيضاء تكوين الأغشية الحيوية في نموذج فأر In Vivo

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

التصور "تشكيل بيوفيلم" في المبيضات البيض باستخدام جهاز موائع جزيئية الآلي

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