העבודה מומן על ידי NIDCD / NIH R01DC003915 אל LO Bakaletz.

1. כינונה biofilm חוץ גופית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מושבות חיידקים (אשר כבר גדל על אגר המתאים לילה), הושעו בתקשורת ואת OD 490 הותאם 0.65 </li><li style=";text-align:right;direction:rtl"> המתלים חיידקים וכתוצאה מכך היה מדולל אז 01:06 (1 ההשעיה מ&quot;ל חיידקי + 5 מ&quot;ל מראש חימם בינוני) ו מודגרות ב 37 ° C עם 5% CO 2 למשך כ 3 שעות כדי להגיע יומן באמצע השלב. </li><li style=";text-align:right;direction:rtl"> לדלל את יומן אמצע ההשעיה 1:2500 עם צמיחה מחומם מראש מדיה מקום 200 μL של דילול לבאר כל שקופית 8-היטב קאמרית. </li><li style=";text-align:right;direction:rtl"> לדגור על 37 מעלות צלזיוס עם 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לאחר כ 16 שעות, בינוני לשאוב מהפינה של החדר כל ולהוסיף 200 μL טרי, מחומם מראש בינוני - לוותר לאורך הקיר של החדר כדי לא ליצור כוחות גזירה בתוך החדר אשר יכול לשבש את biofilm. </li><li style=";text-align:right;direction:rtl"> אם דוגרים יותר מ 24 שעות, שנה בינונית כל 12 שעות או לפי הצורך כדי לשמור על יכולת הקיום חיידקי. </li></ol> 2. ויזואליזציה של biofilm <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב בינוני מתא אחד, כמתואר לעיל, ולשטוף biofilm תושב פעמיים בעדינות עם מלח סטרילית. </li><li style=";text-align:right;direction:rtl"> הוספת 200 μL של (רכיב 3 μL + 3 B מרכיב μL לכל מ&quot;ל של תמיסת מלח) BacLight Live / Dead כתם זה היטב דגירה בטמפרטורת החדר למשך 15 דקות. הגן על תרבות מן האור מנקודה זו ואילך. </li><li style=";text-align:right;direction:rtl"> לשאוב את הכתם ולשטוף פעמיים בעדינות עם תמיסת מלח סטרילית כמו קודם. </li><li style=";text-align:right;direction:rtl"> הוספת 200 μL של נייטרלי שנאגרו פורמלין זה היטב דגירה במשך 30 דקות בטמפרטורת החדר כדי לתקן את הדגימה. </li><li style=";text-align:right;direction:rtl"> הסר מקבע כהלכה להיפטר בהתאם להנחיות מוסדיים. </li><li style=";text-align:right;direction:rtl"> שטפו פעמיים עם מי מלח ועושה את הנוזלים לשטוף המכילים פורמלין לעיל. </li><li style=";text-align:right;direction:rtl"> הסר את בארות פלסטיק משקופית, מוסיפים מלח מספיק זה גם כך שכאשר coverslip מושם על אטם, אין בועות אוויר נוכחים. </li><li style=";text-align:right;direction:rtl"> Coverslip וסוגרים את הקצוות של coverslip עם הרכבה בינונית. לק ניתן להשתמש כדי לאטום את שקופיות שקופיות עם זאת לא יהיה כפי קבע. </li><li style=";text-align:right;direction:rtl"> אפשר איטום להתייבש במשך שעה בערך 1 לפני בחינת דרך מיקרוסקופ. </li></ol> 3. תוצאות <img src="/files/ftp_upload/2481/2481fig1.jpg" alt="איור 1" /> באיור 1. הנציג תמונות מרוכבים 3D של biofilm נוצר שקופית 8-היטב קאמרית. חיידקים תויגו עם כתם חיידקים חי / מת לחיות שבה לזרוח חיידקים ירוקים מת לזרוח אדום. (א) התמונה של Composite biofilm כל הארכיטקטורה נפרדות עם מגדלים ותעלות מים. (ב) biofilm זהה (A), ראיתי עכשיו ב חתך.

<ol>
	<li>Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+otitis+media:+evidence+and+relevance.">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, S17-S19 (2007).</li><li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li></ol>

אין ניגודי אינטרסים הכריז.

הבנת המנגנון שבאמצעותו biofilms הטופס, כמו גם באפיון רכיבי המטריצה ​​שלהם, הוא תחום עניין רב. פרוטוקול זה יכול לשמש כדי לזהות חלבונים או מרכיבים אחרים שתורמים שלמות מבנית של biofilm, וכן לסייע בזיהוי יעד חלבונים אשר עשויים להשאיל את עצמם ביישום הטיפולי. פעמים הדגירה ו דילולים בפרוטוקול לעיל הוקמו ליצירת biofilm אופטימלי על ידי nontypeable Haemophilus influenzae (NTHI). עבודה חלק ראשוני עשוי להיות נחוץ כדי לייעל את הפעולות הבאות עבור מינים חיידקים אחרים (דילול ראשוני כלומר זמן הדגירה הדרושה להשגת שלב אמצע יומן צמיחה). דילול 1:2500 עבור זריעת הראשונית של התאים הוקמה על מנת להבטיח כי biofilm חזקים יפתחו בחדר שקופיות הנגרמת על ידי אורגניזם הזה ובתוך פעמים הדגירה כאמור, ללא biofilm overgrowing החדר ו / או מתיש מזינים זמין . לפיכך, דילול ראשוני או פעמים הדגירה ייתכן שיהיה צורך מותאם זן חיידקים אחרים.

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<td>Chocolate II agar</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>B21267X</td>
<td>&nbsp;</td>
<tr>
<td>8-well chamber slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-656-18</td>
<td>&nbsp;</td>
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<td>BacLight Live/Dead bacterial viability kit</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9439023</td>
<td>&nbsp;</td>
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<td>BHI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>211059</td>
<td>&nbsp;</td>
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<td>Hemin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H5533</td>
<td>&nbsp;</td>
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<td>&beta;-NAD</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N7004</td>
<td>&nbsp;</td>
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<td>Permaslip mounting media</td>
<td>&nbsp;</td>
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<td>NC9693613</td>
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in vitro biofilm formation in an 8-well chamber slide

האופי הכרוני של מחלות רבות מיוחסת היווצרות של biofilms חיידקי אשר הסרבן כדי טיפול אנטיביוטי מסורתי. Biofilms הם הקהילה הקשורים חיידקים מצורף משטח ו עטוף מטריצה. תפקידה של המטריצה ​​תאיים הוא רב פנים, כולל הקלת רכישת מזין, מציעה הגנה משמעותית נגד לחצים סביבתיים (למשל, התגובות מארח החיסונית). במאמץ להשיג הבנה טובה יותר באשר לאופן החיידקים בתוך biofilm להגיב מדגיש הסביבה השתמשנו פרוטוקול שבו אנו לחזות biofilms חיידקים אשר נוצרו בשקופית 8-היטב קאמרית. Biofilms הוכתמו BacLight Live / Dead כתם ובדק באמצעות מיקרוסקופ confocal כדי לאפיין את גודל biofilm יחסית, ומבנה תחת תנאים משתנים הדגירה. Z-מחסנית תמונות נאספו באמצעות מיקרוסקופיה confocal ונותחו על ידי סטטמ&quot;מ. פרוטוקול זה יכול לשמש כדי לעזור להבהיר את מנגנון קינטיקה על ידי היוצרים biofilms, כמו גם לזהות את המרכיבים שחשובים מבנה biofilm ויציבות.

Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

In vitro Biofilm Formation in an 8-well Chamber Slide

מאמר זה מתאר את הליך הקמת ויזואליזציה של biofilm חיידקי גדל בתוך שקופית 8-גם תא

ב גיבוש biofilm חוץ גופית ב 8-Slide גם לשכת

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

in-vitro-biofilm-formation-in-an-8-well-chamber-slide

Research

JoVE Journal

Immunology and Infection

In vitro Biofilm Formation in an 8-well Chamber Slide

39.8K Views.  The Research Institute at Nationwide Children's Hospital. מאמר זה מתאר את הליך הקמת ויזואליזציה של biofilm חיידקי גדל בתוך שקופית 8-גם תא

Video: In vitro Biofilm Formation in an 8-well Chamber Slide

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).

Pseudomonas aeruginosa ו biofilm Saccharomyces cerevisiae בתוך תאים Flow

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilm microtiter תבשיל גיבוש Assay

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

כמותי<em&gt; במבחנה</em&gt; Assay כדי למדוד הידבקות נויטרופילים לתאים מופעלים עיקריים אדם אנדותל כלי הדם בתנאים סטטיים

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

פלישת חיידקי enteric של תאי אפיתל במעי<em&gt; במבחנה</em&gt; באופן דרמטי משופר תוך שימוש במודל לשכת דיפוזיה אנכי

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

מדידת תפוקה גבוהה של שחרור DNA תאי והיווצרות NET כמוני נויטרופילים האנוש<em&gt; במבחנה</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

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.

לדמיין את ההשפעות של כיח על פיתוח biofilm באמצעות מודל תאיים coverglass

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

שיטות ויטרו להשוואת יעד מחייב CDC אינדוקציה בין נוגדנים טיפוליים: יישומים ניתוח Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

An<em&gt; במבחנה</em&gt; Assay מחייב Assay כי חוזה חדירה לסמים בשחפת נגעים

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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.

ויזואליזציה של ביופילמים ב קנדידה אלביקנס שימוש בהתקן Microfluidic אוטומטי

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.

מלח מרה-induced ביופילמים בתוך המעית פתוגנים: טכניקות וכימות

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