بي بفضل كوتشما شيري ، وروبرت بيت نيويل السيقان لتقديم الصور في الشكل 1. وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح R01AI083256 لغاو

1. تنامي وجود بيوفيلم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو ثقافة الزائفة الزنجارية البرية من نوع أو سلالة متحولة بين ليلة في المتوسط ​​الغني (أي LB) </li><li style=";text-align:right;direction:rtl"> تمييع الثقافة في أكثر من ليلة 1:100 متوسطة جديدة لفحوصات بيوفيلم. ومقايسة بيوفيلم القياسية المتوسطة لP. الزنجارية هو M63 المتوسطة تستكمل مع الحد الأدنى من كبريتات المغنسيوم ، والجلوكوز والأحماض casamino (انظر الجدول). كما يمكن الاستعاضة عن بيوفيلم المعززة البديلة المتوسطة التي تحفز نمو أقل العوالق وبيوفيلم أكثر قوة ، والجلوكوز والأحماض casamino مع أرجينين مثل وحيد للكربون ومصدر الطاقة. </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميليلتر من تخفيف لكل بئر 96 في طبق بشكل جيد. لالمقايسات الكمية ، ونحن عادة استخدام الآبار 4-8 تكرار لكل علاج. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة microtiter ل24/04 ساعة عند 37 درجة مئوية. </li></ol> 2. تلطيخ للبيوفيلم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد مرور فترة الحضانة ، تفريغ خارج الخلايا من خلال تحويل لوحة على والرج السائل. </li><li style=";text-align:right;direction:rtl"> بلطف يغرق لوحة صغيرة في حوض من الماء (أي استخدام غيض من مربعات قيعان ماصة للP1000 pipetmen والحوض). نفض الماء. كرر هذه العملية مرة ثانية. هذه الخطوة تساعد على إزالة الخلايا غير مرتبط والمكونات وسائل الاعلام التي يمكن الملون في الخطوة التالية ، ويخفض بشكل كبير تلوين الخلفية. </li><li style=";text-align:right;direction:rtl"> إضافة 125 ميكرولتر من محلول 0.1 ٪ من الكريستال البنفسجي في الماء إلى كل بئر من لوحة microtiter. ارتداء قفازات ومعطف مختبر في حين جعل الحل. الحذر عند استخدام وزنها من السيرة الذاتية كما هو مسحوق hydroscopic البقع بسهولة والملابس ، والجلد ، الخ. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة microtiter في درجة حرارة الغرفة لمدة 10-15 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف لوحة 3-4 مرات مع غمر المياه في حوض من الماء على النحو المبين أعلاه ، ونفض لطخة بقوة على كومة من مناشف ورقية للتخلص من لوحة كل الخلايا الزائدة وصباغة. </li><li style=";text-align:right;direction:rtl"> بدوره لوحة microtiter رأسا على عقب والجافة لبضع ساعات أو طوال الليل. </li><li style=";text-align:right;direction:rtl"> للفحوصات النوعية ، ويمكن تصويرها عند الآبار الجافة. </li></ol> 3. قياس بيوفيلم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 125 ميليلتر من حمض الخل 30 ٪ في كل بئر ماء لوحة من لmicrotiter solubilize السيرة الذاتية. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة microtiter في درجة حرارة الغرفة لمدة 10-15 دقيقة. </li><li style=";text-align:right;direction:rtl"> نقل 125 ميكرولتر من السيرة الذاتية solubilized إلى شقة جديدة طبق microtiter القاع. </li><li style=";text-align:right;direction:rtl"> قياس الامتصاصية في قارئ لوحة عند 550 نانومتر باستخدام حمض الخليك بنسبة 30 ٪ في ماء فارغة. </li></ol> 4. ممثل النتائج : <img src="/files/ftp_upload/2437/2437fig1.jpg" alt="الشكل 1" /> الشكل 1. يظهر نتيجة لممثل فحوصات أجريت لتشكيل بيوفيلم الزائفة الزنجارية ، الزائفة المتألقة والمكورات العنقودية الذهبية. (أ) عرض الجانب من البئر مع بيوفيلم من الزنجارية P. (8 ساعات و 37 درجة مئوية). (ب) وعرض الجانب من البئر مع بيوفيلم من P. المتألقة (6 ساعات و 30 درجة مئوية). (ج) شكلت وجهة نظر من أعلى إلى أسفل من بيوفيلم س. المذهبة في لوحة مسطحة القاع microtiter (بئرين ، 24 ساعة و 37 درجة مئوية). P. والزنجارية P. المتألقة كلاهما الكائنات متحركة وتشكيل بيوفيلم في واجهة الهواء السائل. S. المذهبة وغير متحركة ، وتشكل بيوفيلم في قاع البئر.

<ol>
	<li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol. Microbiol. 28, 449-461 (1998).</li><li>O'Toole, G. A., Doyle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. , 91-109 (1999).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426, 306-310 (2003).</li><li>Kuchma, S. L., Connolly, J. P., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, 1441-1454 (2005).</li><li>Caiazza, N. C., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SadB+is+required+for+the+transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+aeruginosa+PA14.">SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14.</a> J Bacteriol. 186, 4476-4485 (2004).</li><li>Shanks, R. M., Sargent, J. L., Martinez, R. M., Graber, M. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+lock+solutions+influence+staphylococcal+biofilm+formation+on+abiotic+surfaces.">Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.</a> Nephrol Dial Transplant. 21, 2247-2255 (2006).</li><li>Caiazza, N. C., Merritt, J. H., Brothers, K. M., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+regulation+of+biofilm+formation+and+swarming+motility+by+Pseudomonas+aeruginosa+PA14.">Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.</a> J. Bacteriol. 189, 3603-3612 (2007).</li><li>Hinsa, S. M., Espinosa-Urgel, M., Ramos, J. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+fluorescens+WCS365+requires+an+ABC+transporter+and+a+large+secreted+protein.">Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein.</a> Mol Microbiol. 49, 905-918 (2003).</li><li>Mack, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+transposon+mutants+of+biofilm-producing+Staphylococcus+epidermidis+impaired+in+the+accumulative+phase+of+biofilm+production:+genetic+identification+of+a+hexosamine-containing+polysaccharide+intracellular+adhesin.">Characterization of transposon mutants of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intracellular adhesin.</a> Infect. Immun. 62, 3244-3253 (1994).</li><li>Vidal, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Escherichia+coli+K-12+mutant+strain+able+to+form+biofilms+on+inert+surfaces:+involvement+of+a+new+ompR+allele+that+increases+curli+expression.">Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.</a> J. Bacteriol. 180, 2442-2449 (1998).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

يمكن تعديل هذه الطريقة للاستخدام مع طائفة واسعة من الأنواع الجرثومية. الميكروبات متحركة تلتزم عادة على الجدران و / أو قيعان الآبار ، بينما غير متحركة الميكروبات تلتزم عادة إلى أسفل الآبار. يجب تحديد الظروف المثلى لتشكيل بيوفيلم (أي نمو متوسطة ، ودرجة الحرارة ، والوقت...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 X M63</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Prepare as a 5X M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature. Dilute 5X stock 1:5, autoclave, cool, then add the desired components.</td>
</tr>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P285-500</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P288-500</td>
<td>&nbsp;</td>
<tr>
<td>(NH4)2SO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5132</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M63-500</td>
<td>Add to 1 mM final concentration. Prepare as a 1 M stock in water and autoclave.</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>D16-3</td>
<td>Add to 0.2% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Casamino acids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>223050</td>
<td>Add to 0.5% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Arginine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5131</td>
<td>Add to 0.4% final concentration. Prepare as a 20% stock in water and filter sterilize. This alternative carbon/energy source can replace glucose and casamino acids</td>
</tr>
<tr>
<td>Microtiter plates</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353911</td>
<td>Falcon 3911, Microtest III, Flexible assay plates, 96 well, U-bottom, non-sterile, non-tissue-culture treated.</td>
</tr>
<tr>
<td>Microtiter plate lids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353913</td>
<td>The lids can be reused by cleaning with 95% ethanol in water.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>229641000</td>
<td>Prepare as a 0.1% solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

microtiter dish biofilm formation assay

الأغشية الحيوية هي مجتمعات الميكروبات يعلق على الأسطح ، والتي يمكن العثور عليها في المواقع الطبية والصناعية والطبيعية. في الواقع ، الحياة في بيوفيلم ربما يمثل وضع تسود لنمو الميكروبات في معظم البيئات. الأغشية الحيوية الناضجة لها خصائص متميزة قليلة. وتحاط عادة بيوفيلم الميكروبات عن طريق المصفوفة خارج الخلية التي توفر بنية وحماية للمجتمع. الميكروبات تنمو في بيوفيلم أيضا أن يكون سمة العمارة تتألف عموما من macrocolonies (التي تحتوي على آلاف من الخلايا) محاطة مملوءة بسائل القنوات. بيوفيلم نابعة من الميكروبات هي أيضا سيئة السمعة لمقاومتهم لمجموعة من العوامل المضادة للجراثيم بما فيها المضادات الحيوية ذات الصلة سريريا. في مقايسة طبق microtiter أداة هامة لدراسة المراحل المبكرة في تشكيل بيوفيلم ، وأنها طبقت بشكل أساسي لدراسة الأغشية الحيوية البكتيرية ، وعلى الرغم من أنه تم أيضا استخدام هذا الاختبار لدراسة تشكيل بيوفيلم الفطرية. لأن هذا الاختبار يستخدم ثابت ، دفعة للنمو الشروط ، فإنه لا يسمح لتشكيل الأغشية الحيوية الناضجة التي ترتبط عادة مع أنظمة الخلية التدفق. ومع ذلك ، فقد تم فحص فعالية في تحديد العديد من العوامل اللازمة لبدء تشكيل بيوفيلم (أي ، سياط ، الشعرة ، adhesins ، والإنزيمات المشاركة في دوري - DI - GMP ملزمة والأيض) ، وكذلك الجينات المسؤولة عن إنتاج السكاريد خارج الخلية. علاوة على ذلك ، يشير إلى أن الأعمال المنشورة الأغشية الحيوية التي تزرع في أطباق microtiter القيام تطوير بعض خصائص الأغشية الحيوية الناضجة ، مثل التسامح ومقاومة للمضادات الحيوية المستجيبات الجهاز المناعي. هذا الطبق microtiter بسيط يسمح للمقايسة تشكيل بيوفيلم على و / أو الجدار السفلي من صحن microtiter. طبيعة إنتاجية عالية للمقايسة يجعله مفيدا للشاشات الجينية ، وكذلك اختبار تشكيل بيوفيلم عن سلالات متعددة في ظل ظروف النمو المختلفة. وقد استخدمت بدائل من هذا الاختبار في وقت مبكر لتقييم بيوفيلم تشكيل لطائفة واسعة من الميكروبات ، بما في ذلك ولكن ليس على سبيل الحصر ، pseudomonads ، الضمة الكوليرية ، كولاي ، staphylocci ، المعوية ، متفطرات والفطريات. في البروتوكول الموصوفة هنا ، سوف نركز على استخدام هذا الاختبار لدراسة تشكيل بيوفيلم التي الزائفة الزنجارية الحي النموذجي. في هذا الاختبار ، يتم قياس مدى تشكيل بيوفيلم باستخدام صبغة الكريستال البنفسجي (السيرة الذاتية). ومع ذلك ، فقد تم الإبلاغ عن عدد آخر من البقع اللونية والتمثيل الغذائي للالكمي لتشكيل بيوفيلم باستخدام مقايسة لوحة microtiter. حققت سهولة وتكلفة منخفضة ومرونة microtiter مقايسة لوحة أنها أداة حاسمة لدراسة الأغشية الحيوية.

Watch this Scientific Journal Video about Microtiter Dish Biofilm Formation Assay at JoVE.com

Microtiter Dish Biofilm Formation Assay

ويصف وسائل الفحص السريع لقياس وقت مبكر تشكيل بيوفيلم في البكتيريا والفطريات. يستخدم هذا الأسلوب لوحة microtiter كما التحتية لتشكيل بيوفيلم الميكروبية ، وبيوفيلم هو تصور استخدام الكريستال سلالة البنفسجي. في مقايسة يوفر إما فحص نوعي أو كمي لتشكيل بيوفيلم في وقت مبكر.

Microtiter تشكيل بيوفيلم الطبق الفحص

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Research

JoVE Journal

Immunology and Infection

106.9K Views.  Dartmouth Medical School. ويصف وسائل الفحص السريع لقياس وقت مبكر تشكيل بيوفيلم في البكتيريا والفطريات. يستخدم هذا الأسلوب لوحة microtiter كما التحتية لتشكيل بيوفيلم الميكروبية ، وبيوفيلم هو تصور استخدام الكريستال سلالة البنفسجي. في مقايسة يوفر إما فحص نوعي أو كمي لتشكيل بيوفيلم في وقت مبكر.

Video: Microtiter Dish Biofilm Formation Assay

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 ...

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

والزائفة الزنجارية السكيراء بيوفيلم في خلايا خميرة تدفق

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

تشكيل بيوفيلم في المختبر في غرفة الشرائح 8 جيد

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

اختبار الحساسية للمضادات الميكروبات مجمع المتفطرة السل للأدوية الخط الأول والثاني عن طريق التخفيف في شكل حساء بلايت Microtiter

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

فك رموز والتصوير إمراض وحبال من<em&gt; المتفطرة خراج</em&gt; في الزرد الأجنة

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

استهداف بيوفيلم أسوشيتد<em&gt; المكورات العنقودية الذهبية</em&gt; استخدام ريسازورين استنادا الفحص المخدرات-القابلية

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;يبلوع إغلاق الفحص&quot; لتصور يبلوع تشكيل في ثلاثة أبعاد عن طريق التأمل إجمالي الداخلية نيون الميكروسكوب (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

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

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

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

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

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

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

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

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

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

تشكيل بيوفيلم التي يسببها الملح الصفراوية في مسببات الأمراض المعوية: تقنيات لتحديد وتقدير حجم

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

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

مقايسة الفائق تقييم وتقدير حجم تشكيل العَدلات فخ خارج الخلية

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...