Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation - Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

The authors have nothing to disclose.

العصوية الرقيقة أشكال قوية ومنظم للغاية الأغشية الحيوية في كل من السائل (pellicles) وعلى وسط صلب (المستعمرات). وبالتالي، فإنه بمثابة كائن نموذجا مثاليا لوصف طريقة عمل مثبطات بيوفيلم محددة. على وسائل الاعلام الصلبة، الخلايا تشكل هياكل متعددة الخلايا مع السمات المميز?...

المجتمعات البكتيرية متعددة الخلايا تلعب أدوارا هامة في البيئات الطبيعية والبشرية، ويمكن أن تكون مفيدة أو ضارة للغاية. ومن المعروف أن هذه المستعمرات متعددة الخلايا كما الأغشية الحيوية، حيث يتم تضمين الخلايا الفردية في المواد البوليمرية خارج الخلية الذات المنتجة (EPS) المصفوفة. العائد على السهم تلتزم بقوة الخلايا على سطح أنها استعمار. أنها بمثابة درع ضد القوات الميكانيكية والكيميائية وإنشاء اتصال وثيق بين الخلايا المجاورة، وتسهيل الاتصالات الخلوية 1. يمكن مشاهدة بيوفيلم كمجتمع متباينة، حيث تستخدم الخلايا درجة عالية من التنظيم، منسقة عمليات لتنسيق أنشطتها داخل المجتمع، وكذلك عبر الأنواع 2-5. وكثيرا ما يرتبط الانتقال من العوالق، ووضع حرة المعيشة للنمو إلى حالة بيوفيلم مع العمليات التنموية. وخير مثال هو بكتيريا التربة العصوية الرقيقة موجبة الجرام، وبالتالي على undomeيخدم سلالة sticated كما كائن نموذج قوي لدراسة مراحل النمو مما يؤدي إلى تشكيل بيوفيلم. في هذه البكتيريا وخلايا متحركة تنظيم أنفسهم في هياكل متعددة الخلايا واضح لتنفيذ مهام متخصصة 4. مجموعة واحدة من الخلايا، المنتجين مصفوفة تفرز exopolysaccharides 6، بروتين اميلويد الطاسه 7،8، والبروتين للا مائية سطح BslA 9،10. وكلها مشاركة في الجمعية العامة للEPS 11-13. وبالنظر إلى وفرة من الأغشية الحيوية في المنافذ الطبيعية والبشرية والضرر القاتل المفترض أنها يمكن أن تسبب، هناك حاجة ملحة لايجاد سبل لمنع تكونها. يمكن مثبطات جزيء صغير مساعدة في اكتشاف مسارات تنظيمية جديدة، والانزيمات والبروتينات الهيكلية تشارك في تشكيل بيوفيلم، وبالتالي تعزيز الرؤى في العمليات المعقدة التجمع مجتمع متعدد الخلايا. كما B. الرقيقة هو نموذج المدروس لالحيويتشكيل الفيلم 14،15، ويمكن استخدامه لتقييم آثار مختلف مثبطات بيوفيلم. تتناول هذه الدراسة أربعة أساليب الأساسية التي هي مفتاح لتقييم استجابة من الأغشية الحيوية لمثبطات جزيء صغير. أولا، للتأكد من أن هذه المثبطات لديها هدف محدد بيوفيلم، والفصل بين تأثير على نمو العوالق من التأثير على تشكيل بيوفيلم أمر بالغ الأهمية. معظم وكلاء المضادة للبكتيريا تستهدف الخلايا في مرحلة النمو العوالق، ولكن الجزيئات التي تستهدف حياة بيوفيلم نادرة. بالإضافة إلى ذلك، كما الجزيئات التي لا تؤثر على نمو العوالق ليست سامة، يمكن أن تقلل من الضغط الانتقائي لصالح المسوخ المقاومة للمضادات الحيوية 16. على سبيل المثال، عندما يتم التعامل مع الأغشية الحيوية مع الأحماض الأمينية-D أو بعض الجزيئات الأخرى خلية التدخل الجدار، إما بالانزعاج هم أو تفكيكها، ولكن هذه المثبطات فقط تؤثر أقل ما يقال 12،17 نمو العوالق. في المقابل، العديد من المضادات الحيوية يضعف بشكل كبير نمو العوالق، مع لإيتل أو أي تأثير على تشكيل بيوفيلم 17. ثانيا، وضع إطار تجريبي ثابت وقوي لدراسة تأثير الجزيئات الصغيرة أمر بالغ الأهمية. لاحظنا أن نطاق تركيز نشط من مثبطات جزيء صغير حساس للشروط مسبقة الثقافة والإعداد التجريبية المستخدمة لدراسة تأثير هذه المثبطات جزيء صغير. تقارير مختلفة، لا سيما أولئك الذين يدرسون B. الرقيقة، وكشف الاختلافات في مدى التركيز الذي الأحماض الأمينية-D تمنع تشكيل pellicles - العائمة الأغشية الحيوية البكتيرية 12،17-19. وتشير النتائج المعروضة هنا أن العوامل التالية تمثل اختلافات في مدى تركيز نشاط: من شروط مسبقة ثقافة (لوغاريتمي 12،17 مقابل مرحلة نمو 20 في وقت متأخر من ثابتة)، ومتوسط النمو المستخدمة في حالة ما قبل الثقافة (الأغنياء، غير معروف [لوريا مرق، LB] مقابل يعرف [الغلوتامات أحادية الصوديوم-glycerol، MSgg])، ونسبة التلقيح وخصوصا إزالة المتوسطة قبل الثقافة قبل التلقيح. وأظهرت درجة حرارة النمو جليدة ثابت دورا أقل أهمية في نطاق نشاط صغير جزيء المانع D-ليسين، وهو حمض تمثيلي-D الأمينية المستخدمة في هذه الدراسة. وأخيرا، مرة واحدة يتم التعامل مع الأغشية الحيوية مع مثبطات بيوفيلم محددة، يطلب من وسائل قوية ومفيدة لوصف آثار هذه المثبطات على اللياقة البدنية بيوفيلم. هنا، يتم وصف طريقتين لوصف مستقل تأثير مثبطات جزيء صغير بالتفصيل: (1) تأثير على الخلايا وحيدة داخل مستعمرة بيوفيلم ومقاومتها للمضادات الحيوية. الخلايا في الأغشية الحيوية تكون في العادة أكثر مقاومة للمضادات الحيوية بالمقارنة مع البكتيريا التي تعيش خالية من 21-23. رغم أن هذه الظاهرة هي سياقاتها، وغالبا ما تعتبر قدرة EPS للحد من تغلغل المضادات الحيوية كتفسير جذابة 24 </suص&gt;. هذه الطريقة يقيم بقاء خلايا بيوفيلم محددة سلفا بعد التعرض للمواد مضادة للجراثيم. (2) أثر على بنية مستعمرة بيوفيلم، من كبيرة إلى صغيرة الحجم. تتميز المستعمرات بيوفيلم بواسطة هيكلها ثلاثي الأبعاد وجود EPS. باستخدام المجهر الإلكتروني، والتغييرات في شكل الخلية، بنية مستعمرة بيوفيلم والهندسة المعمارية وفرة من EPS يمكن تصور من كبير (ملم) على نطاق صغير (ميكرون).

<table border="0" cellpadding="0" cellspacing="0" width="897">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Luria Broth, Lennox</td>
			<td style="width:305px;">Difco</td>
			<td style="width:127px;">240230</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate monobasic&#160;</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate dibasic&#160;</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher Scientific, 209.27 g/mol</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnesium chloride hexahydrate&#160;</td>
			<td>Merck, 203.30 g/mol&#160;</td>
			<td>1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride anhydrous</td>
			<td>J.T. Baker, 110.98 g/mol</td>
			<td>1311-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manganese(II) chloride tetrahydrate</td>
			<td>Sigma, 197.91 g/mol</td>
			<td>31422-250G-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iron(III) chloride hexahydrate&#160;</td>
			<td>Sigma, 270.30 g/mo)</td>
			<td>F2877-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zinc chloride anhydrous&#160;</td>
			<td>Acros Organics, 136.29 g/mol</td>
			<td>424592500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thiamine hydrochloride</td>
			<td>Sigma, 337.27 g/mol</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-tryptophan</td>
			<td>Fisher Scientific, 204.1 g/mol</td>
			<td>BP395-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-phenylalanine</td>
			<td>Sigma, 165.19 g/mol</td>
			<td>P5482-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-threonine</td>
			<td>Sigma, 119.12 g/mol</td>
			<td>T8625-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol anhydrous</td>
			<td>Bio-Lab Itd</td>
			<td>712022300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamic acid monosodium salts hydrate&#160;</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>G1626-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-leucine</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>855448-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol anhydrous</td>
			<td>Gadot</td>
			<td>830000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">razor blade</td>
			<td>Eddison</td>
			<td>NA</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">circular cellulose filter papers</td>
			<td>Whatman, 90 mm</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutaraldehyde</td>
			<td>EMS (Electron Micoscopy Science), 25% in water</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde&#160;</td>
			<td>EMS, 16% in water</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium cacodylate</td>
			<td>Merck, 214.05 g/mol&#160;</td>
			<td>8.2067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride 2-hydrate</td>
			<td>Merck, 147.02 g/mol&#160;</td>
			<td>1172113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stub-aluminium mount</td>
			<td>EMS, sloted head</td>
			<td>75230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">carbon adhesive tape</td>
			<td>EMS</td>
			<td>77825-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker 37&#176;C</td>
			<td>New Brunswick Scientific Innowa42</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf table top centrifuge 5424</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:301px;">Digital Sonifier, Model 250, used with Double Step Microtip</td>
			<td>Branson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 30 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 23 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter System, 500 ml, polystyrene</td>
			<td>Cornig Incorporated</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotary Shaker - Orbitron Rotatory II</td>
			<td>Boekel</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S150 Sputter Coater&#160;</td>
			<td>Edwards</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPD 030 Critical Point Dryer</td>
			<td>BAL-TEC</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Environmental Scanning Electron Microscope</td>
			<td>XL30 ESEM FEG Philips (FEI)</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

فحص جليدة هو أسلوب واحد لدراسة العمليات درجة عالية من التنظيم والحيوية من B. الرقيقة multicellularity. وبالاضافة الى هذا، هي مناسبة لفحص جليدة لاختبار مجموعة وإما شروط مسبقة بداية أو تركيزات جزيء صغير في لوحة multidish واحدة لزراعة الخلايا في تجربة واحدة. ومع ذلك، B. الرقيق...

Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

منهجيات لدراسة<em&gt; B. الرقيقة</em&gt; الأغشية الحيوية نموذجا للتميز الصغيرة جزيء بيوفيلم المانعون

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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Immunology and Infection

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

15.0K Views.  Weizmann Institute of Science. This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Video: Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

الفئران نموذج تفعيل CD40 - B من الخلايا

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

استخدام نموذج لوصف EpiAirway طويل الأجل المضيف الممرض التفاعلات

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

وحيد الخلية تحليل<emالعصوية الرقيقة> الأغشية الحيوية باستخدام المجهر الإسفار والتدفق الخلوي

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

التهاب القرنية العقبولي المتكررة اللحمية في الفئران، وهذا نموذج لدراسة الإنسان HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

العدوى التجريبية مع<em&gt; المستوحدة الليستيريا</em&gt; نموذجا لدراسة المضيف الردود انترفيرون γ

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

A Murine Model of Group B Streptococcus Vaginal Colonization

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

A Murine Model of Group B Streptococcus Vaginal Colonization

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

نموذج الفئران من المجموعة الثانية<em&gt; العقدية</em&gt; المهبلية الاستعمار

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

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

تصور آثار البلغم للتنمية بيوفيلم باستخدام نموذج غرف ساترة

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

التهابات المفصليات كفحص أدوات للتعرف على العوامل المضادة للفيروسات إيمونومودولاتورس والمضيف الفيروسية

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...