İş JWV grup AB Marie-Curie Reintegration Bursu, Sysmo2 Grant (NWO-ALW/ERASysBio), Horizon hibe (ZonMW) ve Veni dostluk (NWO-ALW) tarafından desteklenmektedir. OPK grubu birkaç STW hibe (NWO), SYSMO1 (IGdeJ) ve SYSMO2 hibe, bir ESF Eurocores SynBio hibe (SynMod) ve Endüstriyel Fermantasyon ve En İyi Gıda ve Beslenme için Enstitüsü Genomik Kluyver Merkezi tarafından desteklenmektedir.

<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>

Çıkar çatışması ilan etti.

, Pek çok diğer tek hücreli tekniklere aksine, burada açıklanan geçen zaman floresan mikroskopi yöntemi atalarının, onun davranış ve bölünme olayları ile ilgili belirli bir hücre tarihini takip kullanılabilir. Floresan etiketli hedef rehberleri veya protein ile birlikte, belirli gelişim süreçleri etkinleştirme süresi ve protein yerelleştirme yanı sıra protein dinamikleri bakteriyel gelişimi sırasında izlenebilir takip edilebilir. Yukarıda belirtildiği gibi, farklı bakteri türleri üzerinde yoğunlaşan çalışmalar, belirli bir bakteri gereklerine göre büyüme koşullarına adapte tarafından yapılabilir. Karşılaştığımız sınırlamalar büyüme koşulları ve örneklem büyüklüğü ile ilgili. Kapalı bir ortam olması nedeniyle, orta koşulları, deneme sırasında değiştirilecek. Ayrıca, deney başına dört suşlarının maksimum verimli bir şekilde takip edilebilir. Bir kaç kritik adımlar göz önüne alındığında, tek bir hücrenin analiz yöntemi, burada açıklanan herhangi bir otomatik mikroskop kullanarak kolayca uygulanabilir. Aşağıda, bu kritik adımların bir bakış verilecektir. Ayrıntılı bilgi ana metin Genel hazırlık: deney öncesinde belirli bir bakteri için gerekli olan otomatik odaklama ayarlarını kontrol etmek akıllıca olacaktır. Aynı şekilde, eğer mümkünse yaklaşık floresan görünüm için en iyi ayarları, önceden belirlenmiş olmalıdır. Ayrıca, hazırlanmış bir zaman çizgisi izleyen zaman içinde kullanıma hazır tüm malzeme (öncesi ısınma, mikroskop odasının, mikroskop ayarları programlama, hücreler istenilen büyüme aşamasında önce bir saat slayt hazırlama, bkz Şekil 1) yardımcı olur B. Büyüme TLM ve CDM subtilis: TLM ve CDM kimyasal açlık medya tanımlanan hangi B. subtilis sadece yavaş yavaş büyür. Hücreleri medya yetiştirilen zaman dilimi belirli bir gerginlik bağlı olarak uzun süreli olabilir. Birbirlerine kazık yavaş büyüme hücreleri engeller mikroskop numunesinin hazırlanması: gen çerçeve, cam slayt ve kapak kayma arasında hava kabarcıkları agaroz tabanlı orta geniş kurumasını önlemek için önlenebilir. Aynı orta / kapak kayma arayüzü için de geçerlidir. Bu hücrelerin yeteri kadar kurumaya bırakın, yüzme ve / veya çok katmanlı büyümesini önlemek için çok önemlidir Time-lapse floresan mikroskopi: Ön ısınma slayt çevre odasının yanı sıra, önemli otofokus sorunları önlemek için çok önemlidir . Bu deney sırasında (örnek yeterince kurutulmuş) alan ve odak kalmak için en yüksek şansı beri Hücreler, bir agar ped ortasında seçili olmalıdır. Deneyler başına 10 yerde maksimum hala düzgün çalışır. Seçildikten sonra ilk hücrenin sadece ilgi odağı ayarlamak için (ayrıntılar için metne bakınız) yazılımını kullanabilirsiniz. Hücreler, ilk saatlerde 30 dakika aralıklarla deney Analiz hala odak olup olmadığını kontrol edin: Bu orta arka plan floresan kanallarda benzer değerlere sahip olup olmadığını önce genişletilmiş analiz prosedürleri kontrol etmek için önemlidir. Sorun Giderme analiz etmek için film zor veya imkansız hale Küçük toz partikülleri, orta bileşenler, kirli lensler veya küçük agaroz kümeleri, yerel olarak artan floresan katkıda bulunabilir: hücreler birbirinin üstüne büyürse, bu da lamel olduğunu gösteriyor olabilir çok erken ya da orta microcolony mono tabakaları büyüme için uygun değildir bağlı. Mutlu slayt diğer hücrelere bölmek ise ilgi hücreler sürekli ölen pozisyonda UV filtre koymak olmadığını kontrol etmek isteyebilirsiniz. Aynı zamanda uzun deneyler sırasında maruz kalma süresi ya da ışık yoğunluğunu azaltmak için yardımcı olabilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Gene Kare </td><td> ABgene </td><td> AB-0578 </td><td> 1.7 x 2.8 cm </td></tr><tr><td> yüksek çözünürlüklü düşük erime agaroz </td><td> Sigma </td><td> A4718 </td><td></td></tr><tr><td> büyük kapak kayma </td><td> birkaç </td><td></td><td> 24 x 50 mm </td></tr><tr><td> istenirse, membran boya, örneğin FM 5-95 </td><td> Invitrogen </td><td> T23360 </td><td> diğer membran boyalar da mevcuttur: <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a> </td></tr><tr><td> Zaman atlamalı mikroskop çevre odasına </td><td> birkaç </td><td></td><td> bizim cihaz için ilgili bölümlerde ayrıntıları görmek </td></tr></tbody></table>

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

Son birkaç yılda bilim adamları mikrobiyal nüfusa dayalı deneylerden elde edilen ortalama veriler tek hücrelerinin davranış, durum veya fenotip temsilcisi olmadığı giderek daha duyarlı hale geldi. Bu yeni anlayış, tek bir hücre çalışmaları sayısı (son değerlendirmeleri için 1,2,3 bakın) sürekli artıyor . Ancak, birçok uygulanan tek bir hücre teknikleri, belirli bir zaman içinde tek bir hücrenin (örneğin, flow sitometri veya standart mikroskopi) gelişimini ve davranışlarını izlemek izin vermez. Burada, biz, takip eden ve kayıt (floresan), Bacillus subtilis ve Streptococcus pneumoniae birçok nesiller için büyüme ve bölünme yoluyla bireysel bakteri hücrelerinin sağlayan birkaç son çalışmalar 4, 5, 6, 7, kullanılan mikroskop yöntemi ayrıntılı bir açıklama sağlar. Ortaya çıkan film, tek bir ortak atadan kökenli bir nüfus içinde tek bir hücrenin geçmişi geri izleme filogenetik soy ağaçları oluşturmak için kullanılabilir. Bu zaman atlamalı floresan mikroskopi yöntemi sadece tek tek hücrelerin büyüme, bölünme ve farklılaşma araştırmak için değil, aynı zamanda hücre geçmişi ve soy spesifik hücresel davranışı üzerinde etkisini analiz etmek için kullanılabilir. Ayrıca, ideal zaman atlamalı mikroskopi, bakteri hücre döngüsü sırasında, gen ekspresyonu dinamikleri ve protein lokalizasyonu incelemek için uygundur. Bu yöntem, bakteri hücrelerinin hazırlamak ve bir microcolony tek hücre akıbet etkinleştirmek için mikroskop lamı inşa nasıl açıklar. Kısacası, tek hücre, büyür ve bir sıcaklık kontrollü çevre oda içinde bir floresan mikroskop altında bölmek agaroz ile desteklenmiş büyüme orta oluşan bir yarı-katı yüzey fark vardır. Görüntüler belirli aralıklarla yakalanır ve daha sonra, açık kaynak yazılımı ImageJ kullanılarak analiz edilmektedir.

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

Bu protokol, otomatik floresan zaman atlamalı mikroskopi kullanılarak zaman farklı bakteriler tek hücre davranışlarını izlemek için bir adım-adım yordam sağlar. Ayrıca, biz nasıl mikroskobu görüntüleri analiz etmek için yönergeler sağlar.

Canlı Hücre Görüntüleme Bacillus subtilis Ve Streptococcus pneumoniae Otomatik Zaman atlamalı Mikroskopi

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

live-cell-imaging-bacillus-subtilis-streptococcus-pneumoniae-using

Research

JoVE Journal

Immunology and Infection

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

42.2K Görüntüleme.  University of Groningen. Bu protokol, otomatik floresan zaman atlamalı mikroskopi kullanılarak zaman farklı bakteriler tek hücre davranışlarını izlemek için bir adım-adım yordam sağlar. Ayrıca, biz nasıl mikroskobu görüntüleri analiz etmek için yönergeler sağlar.

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.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

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

İmmünoloji ve Enfeksiyon

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.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

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 ...

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.

Streptococcus pneumoniae ile İntranazal Kolonizasyon Sırasında İnflamatuar Yanıtların Karakterizasyonu

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

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Etkileşimi incelenmesi<em&gt; Staphylococcus aureus</em&gt; Akım Sitometri ve Time Lapse Mikroskopi tarafından Nötrofiller ile

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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.

Membran Kutup, Membran Dürüstlük ve Hücre içi İyon Konsantrasyonlarında İzleme değişiklikler<em&gt; Streptococcus pneumoniae</em&gt; Floresan boyalar kullanarak

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.

Of kapsül Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Kapsül şişme deneyi kullanarak Reaksiyon

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.

Kapsüler Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Lateks Aglutinasyon tarafından

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.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

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.

Tam Floresans Zaman atlamalı Görüntüleme<em&gt; S. venezuelae</emBir mikroakışkan Cihaz Kullanımı&gt; Yaşam Döngüsü

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

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.