עבודה בקבוצה של JWV נתמך על ידי מארי קירי של האיחוד האירופי Reintegration המלגה, גרנט Sysmo2 (NWO-ALW/ERASysBio), אופק מענק (ZonMW) ועל ידי מלגה VENI (NWO-ALW). הקבוצה של OPK נתמכת על ידי מספר מענקים STW (NWO), SYSMO1 (IGdeJ) ו SYSMO2 מענק, Eurocores ESF SynBio מענק (SynMod) ועל ידי המרכז Kluyver עבור Genomics של תסיסה תעשייתי במכון מובילות המזון והתזונה.

<ol>
	<li>Veening, J. W., Smits, W. K., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability,+epigenetics,+and+bet-hedging+in+bacteria.">Bistability, epigenetics, and bet-hedging in bacteria.</a> Annu. Rev Microbiol. 62, 193-193 (2008).</li><li>Dubnau, D., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability+in+bacteria.">Bistability in bacteria.</a> Mol Microbiol. 61, 564-564 (2006).</li><li>Locke, J. C., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+movies+to+analyse+gene+circuit+dynamics+in+single+cells.">Using movies to analyse gene circuit dynamics in single cells.</a> Nat. Rev. Microbiol. 7 (5), 383-383 (2009).</li><li>Veening, J. 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W., Murray, H., Errington, 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+mechanism+for+cell+cycle+regulation+of+sporulation+initiation+in+Bacillus+subtilis.">A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis.</a> Genes Dev. 23 (16), 1959-1959 (2009).</li><li>Eberhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+localization+of+choline-utilization+proteins+in+Streptococcus+pneumoniae+using+novel+fluorescent+reporter+systems.">Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems.</a> Mol Microbiol. 74 (2), 395-395 (2009).</li><li>Vasantha, N., Freese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+changes+during+Bacillus+subtilis+sporulation+caused+by+deprivation+of+guanine+nucleotides.">Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides.</a> J Bacteriol. 144 (3), 1119-1119 (1980).</li><li>Stewart, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+death+in+an+organism+that+reproduces+by+morphologically+symmetric+division.">Aging and death in an organism that reproduces by morphologically symmetric division.</a> PLoS. Biol. 3, 45-45 (2005).</li><li>Rosenfeld, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+at+the+single-cell+level.">Gene regulation at the single-cell level.</a> Science. 307 (5717), 1962-1962 (2005).</li><li>Guberman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSICIC:+noise+and+asymmetry+in+bacterial+division+revealed+by+computational+image+analysis+at+sub-pixel+resolution.">PSICIC: noise and asymmetry in bacterial division revealed by computational image analysis at sub-pixel resolution.</a> PLoS. Comput. Biol. 4 (11), 1000233-1000233 (2008).</li><li>Montero, L. l. o. p. i. s. . P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+the+flow+of+genetic+information+in+bacteria.">Spatial organization of the flow of genetic information in bacteria.</a> Nature. 466, 77-77 (2010).</li><li>Avery, O. T., Macleod, C. M., McCarty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+chemical+nature+of+the+substance+inducing+transformation+of+pneumococcal+types:+Induction+of+transformation+by+a+desoxyribonucleic+acid+fraction+isolated+from+pneumococcus+type+III.">Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III.</a> J Exp. Med. 79, 137-137 (1944).</li><li>Hoskins, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+of+the+bacterium+Streptococcus+pneumoniae+strain+R6.">Genome of the bacterium Streptococcus pneumoniae strain R6.</a> J Bacteriol. 183, 5709-5709 (2001).</li><li>Martin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recA+gene+of+Streptococcus+pneumoniae+is+part+of+a+competence-induced+operon+and+controls+lysogenic+induction.">The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction.</a> Mol Microbiol. 15, 367-367 (1995).</li></ol>

אין ניגודי אינטרסים הכריז.

בניגוד אחרים טכניקות רבות תא בודד, בשיטה זמן לשגות מיקרוסקופ פלואורסצנטי המתואר כאן ניתן להשתמש כדי לעקוב אחר ההיסטוריה של תא ספציפי לגבי אבותיה, התנהגותה, אירועים חלוקה. בשילוב עם היזמים היעד או חלבונים שכותרתו fluorescently, מסלול ספציפי ההפעלה התפתחותית ניתן בעקבות בזמן ולוקליזציה חלבון, כמו גם הדינמיקה חלבון יכול להיות פיקוח במהלך התפתחות חיידקים. כפי שצוין לעיל, מחקרים להתרכז מיני חיידקים שונים ניתן לבצע על ידי התאמת תנאי הגידול בהתאם לדרישות עבור חיידק ספציפי. המגבלות רק נתקלנו קשורות התנאים לצמיחה גודל המדגם. בשל הסביבה אטום, התנאים בינוני לא ניתן לשנות במהלך הניסוי. יתר על כן, לכל היותר ארבעה זנים לכל ניסוי ניתן לפקח ביעילות. בהתחשב כמה צעדים קריטיים, שיטת הניתוח תא יחיד המתואר כאן יכול בקלות להיות מיושם באמצעות כל מיקרוסקופ אוטומטיות. בחלק הבא, סקירה של השלבים הקריטיים תינתן. מידע מפורט ניתן למצוא את הטקסט העיקרי הכנה כללי:. רצוי לבדוק את הגדרות פוקוס אוטומטי נדרש חיידק ספציפי לפני הניסוי. כמו כן, הגדרות אופטימליות המשוער עבור להדמיה של הקרינה יש לקבוע מראש, אם אפשר. יתר על כן, לאחר שהוכן קו הזמן עוזר לקבל את כל החומר מוכן לשימוש בזמן (לפני ההתחממות קאמרית מיקרוסקופ, תכנות ההגדרות מיקרוסקופ, בהכנת שעה שקופית אחת לפני התאים נמצאים בשלב הצמיחה הרצוי, ראה תרשים 1) . הצמיחה של B. subtilis ב TLM ו CDM: TLM ו CDM מוגדרים כימי התקשורת הרעבה שבו B. subtilis רק גדל לאט. זמן התקופה שבה התאים גדלים בתקשורת עלול להיות ממושך בהתאם לזן הספציפי. הצמיחה האיטי מונע תאים נערמים אחד על השני הכנת המדגם מיקרוסקופ:. בועות אוויר בין מסגרת הגן, את השקופית זכוכית לכסות את הפתק צריך להיות מונע על מנת למנוע ייבוש נרחב של המדיום agarose מבוסס. הוא הדין ממשק בינוני / לכסות את הפתק. זה חיוני כדי לאפשר לתאים יבש מספיק, כדי למנוע שחייה ו / או צמיחה שכבת מרובים זמן לשגות מיקרוסקופ פלואורסצנטי:. טרום ההתחממות של השקופית, כמו גם את חדר הסביבה הוא חיוני כדי למנוע בעיות פוקוס מרכזי. תאים יש לבחור באמצע כרית אגר, שכן אלה יש את הסיכוי הגבוה ביותר להישאר בתחום ולהתמקד במהלך הניסוי (בתנאי המדגם היה יבש מספיק טוב). עד 10 מקומות לכל הניסויים עדיין עובד כמו שצריך. לאחר שנבחר על התא הראשון עניין רק להשתמש בתוכנה כדי לכוון את המיקוד (ראה טקסט לפרטים נוספים). בדוק אם התאים הם עדיין להתמקד במהלך השעות הראשונות של הניסוי ב 30 דקות במרווחים ניתוח:. חשוב לבדוק לפני ניתוח ההליכים הוארכה אם הרקע של המדיום יש ערכים דומים בערוצים פלואורסצנטי. חלקיקי אבק קטנים, רכיבים בינוני, עדשות מלוכלך או גושים זעירים agarose יכול לתרום הקרינה גדל באופן מקומי, מה שהופך את הסרט קשה או בלתי אפשרי לנתח בעיות הירי:. אם התאים לגדול על גבי אחד את השני, זה עלול גם להצביע על כך coverslip היה מצורף מוקדם מדי או כי המדיום אינו מתאים לצמיחה של monolayers microcolony. אם התאים עניין ברציפות למות בטרם עת, ואילו בתאים אחרים בשקופית לחלק בשמחה, אולי כדאי לך לבדוק אם אתה שם את UV-מסנן במצב. זה יכול גם לעזור כדי להקטין את זמן החשיפה או עוצמת האור במהלך הניסויים הארוך.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> ג&#39;ין מסגרת </td><td> ABgene </td><td> AB-0578 </td><td> 1.7 x 2.8 ס&quot;מ </td></tr><tr><td> רזולוציה גבוהה נמוך ההיתוך agarose </td><td> סיגמא </td><td> A4718 </td><td></td></tr><tr><td> מכסה גדול להחליק </td><td> מספר </td><td></td><td> 24 x 50 מ&quot;מ </td></tr><tr><td> אם רוצים, קרום צבע, למשל FM 5-95 </td><td> Invitrogen </td><td> T23360 </td><td> צבעים קרום אחרים זמינים גם: <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a> </td></tr><tr><td> זמן לשגות מיקרוסקופ עם תא סביבתי </td><td> מספר </td><td></td><td> לראות את הפרטים של המכשיר שלנו בסעיפים המתאימים </td></tr></tbody></table>

live cell imaging of bacillus subtilis and streptococcus pneumoniae using automated time-lapse microscopy

במהלך השנים האחרונות הפך מדענים מודעים יותר ויותר כי נתונים שהתקבלו בניסויים הממוצע באוכלוסייה חיידקים מבוסס אינם נציג מעמד התנהגות, או הפנוטיפ של תאים בודדים. בעקבות תובנה חדשה זו מספר מחקרים תא בודד עולה בהתמדה (עבור הסקירות האחרונות לראות 1,2,3). עם זאת, רבים של טכניקות תא בודד להחיל אינם מאפשרים מעקב אחר התפתחות והתנהגות של תא אחד בודד מסוימת בזמן (cytometry זרימת למשל או במיקרוסקופ רגיל). כאן, אנו מספקים תיאור מפורט של שיטה מיקרוסקופית שימוש במחקרים האחרונים מספר 4, 5, 6, 7, אשר מאפשר הקלטה הבאים (הקרינה של) תאים חיידקיים בודדים של Bacillus subtilis ו Streptococcus pneumoniae באמצעות צמיחה וחלוקת במשך דורות רבים. הסרטים וכתוצאה מכך ניתן להשתמש כדי לבנות שושלת עצים פילוגנטי על ידי התחקות בחזרה את ההיסטוריה של תא בודד בתוך אוכלוסיה שמקורם מאב קדמון משותף אחד. זמן לשגות זה מיקרוסקופ פלואורסצנטי השיטה יכולה לשמש לא רק לחקור, צמיחה חלוקה והתמיינות של תאים בודדים, אלא גם לנתח את ההשפעה של ההיסטוריה התא ממוצא על התנהגות סלולרית ספציפית. יתרה מכך, זמן לשגות מיקרוסקופ הוא אידיאלי לבחון ביטוי גנים הדינמיקה לוקליזציה חלבון במהלך מחזור תא החיידק. השיטה מסביר כיצד להכין את תאים חיידקיים ולבנות את השקופית מיקרוסקופ כדי לאפשר את תולדה של תאים בודדים לתוך microcolony. בקיצור, תאים בודדים הם הבחינו על משטח מוצק למחצה המורכב בינוני צמיחה בתוספת agarose שבו הם גדלים ומתחלקים תחת מיקרוסקופ פלואורסצנטי בתוך חדר מבוקר טמפרטורה סביבתיים. תמונות נלכדים במרווחים ספציפיים מנותחים מאוחר יותר בעזרת תוכנות קוד פתוח ImageJ.

Watch this Scientific Journal Video about Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy at JoVE.com

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

פרוטוקול זה מספק הליך צעד אחר צעד כדי לפקח על התנהגות תא בודד של חיידקים שונים בתוך זמן באמצעות מיקרוסקופ פלואורסצנטי אוטומטיות זמן לשגות. יתר על כן, אנו מספקים הנחיות כיצד לנתח את תמונות מיקרוסקופ.

תא הדמיה חיה של Bacillus subtilis ו Streptococcus pneumoniae באמצעות מיקרוסקופית אוטומטי זמן לשגות

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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JoVE Journal

Immunology and Infection

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

42.2K Views.  University of Groningen. פרוטוקול זה מספק הליך צעד אחר צעד כדי לפקח על התנהגות תא בודד של חיידקים שונים בתוך זמן באמצעות מיקרוסקופ פלואורסצנטי אוטומטיות זמן לשגות. יתר על כן, אנו מספקים הנחיות כיצד לנתח את תמונות מיקרוסקופ.

Video: Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

באמצעות הדמיה Bioluminescent לחקור סינרגיזם בין Streptococcus pneumoniae ו שפעת וירוס עכברים תינוקות

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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.

מתא בודד ניתוח בצילוס subtilis Biofilms באמצעות מיקרוסקופ פלואורסצנטי ו cytometry זרימה

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 ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ויזואליזציה של תגובות מושרות רעלן של חיידקים באמצעות מיקרוסקופ פלואורסצנטי תא החי

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

מיקרוסקופיה וידאו חי מתא phagocytosis הפתוגן פטרייתיים

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

אפיון של תגובות דלקתיות במהלך קולוניזציה תוך-אפית עם דלקת ריאות סטרפטוקוקוס

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

ניטור שינויים בממברנה קוטביות, ממברנה יושרה, וריכוזי יון תאיים ב<em&gt; Streptococcus pneumoniae</em&gt; שימוש צבעי ניאון

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

קופסית Serotyping של<em&gt; Streptococcus pneumoniae</em&gt; שימוש תגובת Quellung

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

קופסית Serotyping של<em&gt; Streptococcus pneumoniae</em&gt; על ידי לאטקס התלכדות

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

מדידת תגובות פיזיולוגיות של<em&gt; תסיסנית</em&gt; עצב סנסורי כדי ליפידים פרומונים שימוש סידן הדמיה חיה

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

זמן לשגות הדמיה הקרינה של השלם<em&gt; S. venezuelae</em&gt; מחזור החיים באמצעות מכשיר microfluidic

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...