Diese Arbeit wurde von der Bill-und-Melinda-Gates-Stiftung (PneuCarriage Projekt, Grant 52099) und operativen Infrastruktur-Support-Programm der Regierung von Victoria unterstützt. Dank Eileen Dunne, Janet Strachan, und Kim Hasen für die kritische Durchsicht des Manuskripts.

1. Herstellung von Zellsuspension Typing <ol><li> Mit einer sterilen Einweg-1 ul-Schleife, einen kleinen Schwung der frischen Übernachtreinkultur von Pneumokokken auf festen nichtselektiven Pferdeblut-Agar und impfen 100 ul Heart Infusion (HALLO) Brühe. Die Suspension sollte zu sein scheinen nur sichtbar trüb. Rühren Sie vorsichtig das Rohr zu emulgieren. Hinweis: Andere Medien, wie Hirn-Herz-Infusionsbrühe kann ebenfalls geeignet sein. Zugabe von Serum (z. B. eine Endkonzentration von 10% (v / v) Pferdeserum) und Inkubation bei 37 ° C für ungefähr 1 Stunde vor der Verwendung verwendet werden, um die Reaktion zu verbessern. </li></ol> 2. Überprüfen Dichte der Zellsuspension Die Zellsuspension in Schritt 1.1 hergestellten auch als negative Kontrolle verwendet werden, und sollte für die Zelldichte überprüft werden, bevor die Eingabe Seren. <ol><li> Mit einem 1 ul Schleife übertragen einen Tropfen des Inokulums in Schritt 1.1 vorbereitet auf ein Glass Objektträger. </li><li> Mit einer Pinzette, legen Sie einen kleinen kreisförmigen Deckglas auf der Suspension. </li><li> Mit Phasenkontrasteinstellung, beobachten die Vorbereitung mikroskopisch unter 400-facher Vergrößerung, um die Dichte der Suspension (Abbildung 1A) zu überprüfen. <ol><li> Wenn der Impfstoff ist zu schwer (2A), fügen Sie mehr Brühe zu der Zellsuspension in Schritt 1.1 zu verdünnen gemacht. Vorsichtig wieder mischen. </li><li> Wenn das Inokulum zu gering ist, gibt es beispielsweise bei &lt;50 Zellen sichtbar im mikroskopischen Bereich (2B), fügen mehr Bakterien zur Herstellung der Zellsuspension in Schritt 1.1 hergestellt und vorsichtig mischen. Hinweis: Mit der Erfahrung kann die Dichte der Zellsuspension durch Schütteln und hält sie gegen das Licht, wo es zu sein scheinen nur sichtbar trübe beurteilt werden. </li></ol></li><li> Wiederholen Sie die Schritte 2.1-2.3, bis eine geeignete Anzahl von Zellen in der mikroskopischen Feld sichtbar sind. Hinweis: Glasdias und coverslips präsentieren eine scharfe Gegenstände Gefahr. Nach Gebrauch werden die Objektträger und Deckgläser mit infektiösem Material kontaminiert und muss in ein Biohazard scharfen Container entsorgt werden. </li></ol> 3. Typing <ol><li> Übertragen ~ 1 ul der vorbereiteten Inokulums (zB mit einem 1 ul-Schleife) auf die Glasobjektträger. Entsorgen Sie die Schleife in den Biohazard-Behälter für scharfe Gegenstände. </li><li> Gleiches Volumen (1 ul Öse) von Raumtemperatur-Antiserum auf den Objektträger und mischen beide fällt auch mit der Schleife. </li><li> Mit einer Pinzette, legen Sie einen kleinen kreisförmigen Deckglas auf die Suspension, kümmert das Drop nicht zu berühren. Platzieren des Deckglases auf die Suspension unmittelbar nach der Zugabe von Antiseren, um die Flüssigkeit auf der Folie vor dem Austrocknen zu verhindern. </li><li> Unter dem Mikroskop beobachten die Suspension unter Phasenkontrast mit 400-facher Vergrößerung. Eine negative Reaktion Quellung beobachtet wird, wenn wenig oder kein &quot;Schwellung&quot; der Pneumokokken-Zellen bei 40 gesehen werden0X Vergrößerung, ähnlich dem, was in der Pneumokokken-Zellsuspension ohne Antiseren beobachtet hinzugefügt (Negativkontrolle). Eine positive Reaktion Quellung wird beobachtet, wenn die Zellen im Vergleich zu den Zellen in der Negativkontrolle erscheint vergrößert oder &quot;geschwollen&quot; und mehr sichtbar. Hinweis: Nicht eindeutige Reaktionen sollten durch Untersuchen der Reaktion nach 5-10 min Inkubation bei Raumtemperatur, oder durch Wiederholen der Reaktion mit Antiserum, weiter untersucht werden, zum Beispiel. </li><li> Führen Pneumokokken Serotypisierung mit aufeinanderfolgenden Prüfung der einzelnen Antiseren Pools, bis eine positive Reaktion beobachtet. Alternativ können Pools in Abhängigkeit von lokalen Auftreten von Serotypen &quot;Runden&quot; angewendet werden. Hinweis: Die Prüfung in &quot;Runden&quot; sorgt dafür, dass die Pools mit Antikörpern gegen die häufigsten Serotypen werden zunächst getestet, minimiert Aufwand und Kosten. Gleichzeitige Prüfung von mehreren Pools bietet auch zusätzliche Negativkontrollen, die in interp nützlich sein kann,retation. Wenn alle Pools in einem bestimmten &quot;runde&quot; negativ sind, testen Sie das Isolat mit folgenden Runden der Schwimmbäder. </li><li> Testen Sie das Isolat mit der entsprechenden Muster-oder Gruppenantiserum innerhalb des reaktiven Pool enthalten. Verwenden Sie gegebenenfalls Faktor Antiseren gegen den Serotyp weiter zu differenzieren. Hinweis: Schlüssel zur Pneumokokken-Antiserum vom Hersteller geliefert werden, bei der Auswahl der entsprechenden Pool, Typ, Gruppe oder Faktor Antiseren und Interpretation der Ergebnisse (einschließlich Kreuzreaktionen) eingesetzt. </li><li> Wenn negative Ergebnisse werden mit allen Pools erhalten, testen Sie die Isolat mit Omniserum Reagenz (enthält Antikörper gegen 91 Serotypen 21). Wenn eine positive Reaktion Quellung noch nicht beobachtet, wenn die Prüfung ein Pneumokokken-Isolat mit der Omniserum Reagenz kann dies daran liegen, das Isolat exprimieren geringe oder keine Kapsel oder dass Antikörper gegen die Serotypen sind nicht im Omniserum dargestellt. Die letztere Kategorie können neue Serotypen enthalten. </li> &lt;/ Ol&gt;

<ol>
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Dis. 4, 144-154 (2004).</li><li>Henrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+newly+recognized+types+of+Streptococcus+pneumoniae.">Six newly recognized types of Streptococcus pneumoniae.</a> J. Clin. Microbiol. 33, 2759-2762 (1995).</li><li>Calix, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical,+genetic,+and+serological+characterization+of+two+capsule+subtypes+among+Streptococcus+pneumoniae+Serotype+20+strains:+discovery+of+a+new+pneumococcal+serotype.">Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype.</a> J. Biol. Chem. 287, 27885-27894 (2012).</li><li>Hausdorff, W. P., Bryant, J., Paradiso, P. R., Siber, G. 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Die Autoren haben nichts zu offenbaren.

Ein kritischer Schritt in diesem Verfahren ist eine reine Kultur sicherzustellen, wird die Suspension zusammen mit dem Hinzufügen einer geeigneten Anzahl von Bakterienzellen, in die Brühe herzustellen. Es sei darauf hingewiesen, dass das Wachstum und Koloniemorphologie auf Agarplatten variiert zwischen verschiedenen Pneumokokken-Isolaten werden. Einige produzieren trockene und raue Kolonien, die schwierig sein kann, zu holen mit einer Schleife und nicht gut in der Brühe zu emulgieren. Aus diesem Grund kann eine größere Schleife der Bakterien benötigt werden bei der Herstellung der Zellsuspension. In jedem Fall ist es unerlässlich, dass die einzelnen Zellen und keine Aggregate sind in der Negativkontrolle offensichtlich so ihre relative Größe der Zellen mit Antiseren verglichen werden zugegeben. Zusätzlich können zu viele Bakterienzellen in einem falsch-negativen Reaktion, in dem ein Überschuss von Kapsel-Antigen kann die Quellung Reaktion 10 hemmen führen. Aus diesem Grund ist es wichtig, eine Bakteriensuspension mit geeigneter Zelldichte (Fig. 1) zu schaffen. Die susRente kann für mehrere Monate durch die Zugabe von einigen Tropfen konzentrierter Formalin konserviert werden. Dies ermöglicht eine einfachere Chargenprüfung und sterilisiert die Brühe reduziert das Risiko von Laborinfektionen, obwohl dies muss vor dem Sicherheitsrisiko im Umgang mit Formalin, die giftig, ätzend, krebserzeugend, sensibilisierend und entflammbar ist abgewogen werden. Außerdem haben wir beobachtet, dass einige Isolate zeigen schwächere Reaktionen in Gegenwart von Formalin (Daten nicht gezeigt). Quellung Reaktionen werden unter Verwendung eines Mikroskops untersucht. Die Verwendung von Phasenkontrast verbessert die Sichtbarkeit der Kapsel. In einigen Labors wird die Reaktion unter Verwendung von Ölimmersions bei 1000-facher Vergrößerung 14, 16 betrachtet. Darüber hinaus sind einige Laboratorien eine Gegenfärbung (Methylenblau), um die Sichtbarkeit einer positiven Reaktion 10, 16, 22 weiter zu verbessern. Die hier vorgestellte Methode wird ohne Öl-i angesehenmmersion und unter 400-facher Vergrößerung 15, 17. Diese Methode ist schnell, einfach und relativ leicht zu lesen 23. Es ist wichtig, gute interne Qualitätskontrolle zu halten, um sicherzustellen, dass die Antisera und Testverfahren sind in Ordnung. Die Teilnahme an einer externen Qualitätssicherungsprogramm ist oft von Vorteil. Eine wesentliche Einschränkung der Quellung Reaktion zur Serotypisierung von Pneumokokken sind die hohen Kosten der benötigten Antiseren. Dies wird jedoch in dem obigen Protokoll aufgrund der geringen Menge von Antiserum für jede Reaktion verwendet wird, minimiert. Mehrere andere Verfahren wurden zur Serotypisierung Pneumokokken 23, 24 beschrieben. Allerdings bleibt Quellung der Goldstandard und die Entwicklung von neuen Methoden erfordert Standardisierung gegen die Quellung Reaktion 23. Mit entsprechenden Antiseren kann die Quellung Reaktion auch genutzt werden, um zu identifizieren, und geben Sie anderen Kappen werdenModul 25 produzierenden Bakterien. Die Quellung Reaktion ist maßgeblich an der Charakterisierung der aktuellen und zukünftigen Pneumokokken-Serotypen, sowie Bereitstellung von Informationen über die Wirksamkeit des Impfstoffes und die langfristigen Auswirkungen der Impfung auf Schlitten und Krankheit.

Streptococcus pneumoniae (Pneumokokken) ist für den Tod von schätzungsweise 800.000 Kinder unter fünf Jahren pro Jahr verantwortlich, wobei die Last der Krankheit vor allem in ressourcenarmen Ländern 1 fällt. Pneumokokken-Erkrankungen reichen im Schweregrad von lokalisierten Infektionen wie akute Mittelohrentzündung zu schweren lebensbedrohlichen Infektionen wie Sepsis, Meningitis und Lungenentzündung 2. Über 90 Serotypen von Pneumokokken-Kapselpolysaccharid beschrieben worden, 3, 4. Strom Pneumokokken-Impfstoffe einen Schutz gegen die Serotypen, die für die Mehrzahl der invasiven Erkrankung 5, 6 verantwortlich sind. Allerdings hat weit verbreiteten Einsatz von Impfstoffen mit dem Ersatz der Serotypen, die im Impfstoff enthaltenen Serotypen von nonvaccine in Verbindung gebracht, sowohl im Nasen-Rachen-Wagen und invasive Erkrankungen 7, 8. Dies unterstreicht die Bedeutung der Serotypisierung für epidemiologischeÜberwachungs-und langfristigen Auswirkungen Impfstoffstudien 9. Die Goldstandardmethode für die Pneumokokken-Typisierung ist der Kapselreaktionstest, da die Quellung Reaktion bekannt, die zuerst von Neufeld im Jahre 1902 beschrieben (siehe österreichischen 10). Ein hohes Maß an Übereinstimmung zwischen Laboratorien und zwischen der Quellung Reaktion und andere Methoden Serotypisierung 11-13 gefunden. Es gibt mehrere Varianten der Quellung Reaktion 10, 14-16. Das hier beschriebene Verfahren erfordert nicht die Verwendung einer Gegenfärbung oder der Ölimmersionslinse, sondern es wird ein Tropfen vorbereiteten Probe gemischt mit dem Eingabe-Serum untersucht und direkt unter 400-facher Vergrößerung. Die Quellung Reaktion wird üblicherweise unter Verwendung von handelsüblichen Antisera. Das hier beschriebene Verfahren verwendet weniger Antiseren verglichen mit anderen Methoden, 16, 17, so dass es kostengünstiger. Sobald ein isol.gegessen wird als Pneumokokken identifiziert, wird es nacheinander mit Antiseren Pools getestet, bis eine positive Reaktion beobachtet wird. Jeder Pool Antiserum enthält verschiedene Mischungen von Antiseren gegen 91 Pneumokokken-Serotypen 18 angehoben. Sobald der Pool aufgebaut ist, werden die einzelnen Muster und Gruppen Antiseren, die im reaktiven Pool aufgenommen werden einzeln nacheinander getestet. Typ Antiseren reagieren in der Regel mit einem einzigen Serotyp (zB Typ-1-Antiserum mit Serotyp 1 reagiert), während die Gruppe Antiseren reagieren mit allen Serotypen, die in einer bestimmten Gruppe (zB Gruppe 22 Antiserum reagiert mit Serotypen 22F und 22A). Einige Serotypen innerhalb der Gruppen werden weiter mit Faktor Antiseren (zB Serotyp 22F reagiert mit Faktor 22b, aber nicht 22c) unterschieden. In diesem Fall ist die Prüfung erfolgt mit allen relevanten Faktor Antiseren fortgesetzt, bis Serotyp bestimmt 19, 20. Die Quellung Reaktion ist einfach und schnell 14 und kann in jeder l durchgeführt werdenabor ist, dass mit einer guten Qualität Mikroskop und entsprechenden Satz von Antiseren ausgestattet.

<table border="0" cellpadding="0" cellspacing="0" width="1305">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Neufeld) antisera</td>
			<td>SSI Diagnostica</td>
			<td></td>
			<td><a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a> 
			Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Omni) antiserum</td>
			<td>SSI Diagnostica</td>
			<td>2438</td>
			<td>Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Microscope</td>
			<td>Leica Microsystems</td>
			<td>Leica DML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated Disposable Inoculating Loops 1 &#181;l</td>
			<td>Copan</td>
			<td>CD175S01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse Blood Agar (HBA) plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>PP2001</td>
			<td><a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au/</a> 
			Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heart Infusion (HI) broth</td>
			<td>Media Preparation Unit (MPU), The University of Melbourne</td>
			<td>40HIN</td>
			<td>Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides 76 mm x 26 mm</td>
			<td>Lomb Scientific</td>
			<td>7101</td>
			<td>Sharps hazard: Dispose in a biohazard sharps container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips</td>
			<td>NeuVitro&#160;</td>
			<td>GG-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">*Excluding the antisera, all other materials can be purchased from alternative suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Plates prepared from defibranated sheep blood are suitable but blood plates prepared from citrated blood or human blood are not</td>
		</tr>
	</tbody>
</table>

capsular serotyping of streptococcus pneumoniae using the quellung reaction

Es gibt über 90 verschiedene Kapsel Serotypen von Streptococcus pneumoniae (Pneumokokken). Abgesehen davon, dass ein Werkzeug für das Verständnis Pneumokokken-Epidemiologie, können Kapsel Serotypisierung nützliche Informationen für die Impfstoff-Wirksamkeit und Wirkungsstudien liefern. Die Quellung Reaktion ist der Goldstandard-Methode für Pneumokokken-Kapsel Serotypisierung. Das Verfahren beinhaltet das Testen einer Pneumokokken-Zellsuspension mit gepoolten und spezifische Antiseren gegen die Kapsel-Polysaccharid gerichtet. Die Antigen-Antikörper-Reaktionen werden mikroskopisch beobachtet. Das Protokoll besteht aus drei Schritten: 1) Herstellung einer Bakterienzellsuspension, 2) Mischen der Zellen und Antiseren auf einem Glasträger, und 3) das Lesen der Quellung Reaktion unter Verwendung eines Mikroskops. Die Quellung Reaktion ist relativ einfach durchzuführen und kann angewendet werden, wo ein geeignetes Mikroskop und Antiseren zur Verfügung.

<html> Eine positive Reaktion tritt auf, wenn Quellung typspezifischen Antikörper bindet an die Kapsel der Pneumokokken, die zu einer Änderung seines Brechungsindex 10. Wenn unter einem Mikroskop betrachtet, erscheinen die Bakterien &quot;geschwollen&quot; und mehr sichtbar 14. 1A zeigt Pneumokokken-Zellen in HALLO Brühe. Bei Typ-spezifische Antiserum wird in die Bakteriensuspension hinzugefügt, Pneumokokken erscheinen &quot;geschwollen&quot;, refraktive und mehr abgerundet (Abbildung 1B). Zu viele Bakterienzellen in die Brühe zugegeben wird, kann in Aggregation von Zellen oder einem falsch-negativen Reaktion führen. Abbildung 2 zeigt Inokula der Pneumokokken-Zellen in HALLO Brühe, die &quot;zu schwer&quot; (2A) oder &quot;zu leicht&quot; (2B) sind. 08fig1highres.jpg &quot;src =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; Fig. 1 ist. Negative und positive Quellung Reaktion. Zubereitungen von der Serotyp 9V Pneumokokken isolieren, ohne Antiserum (1A) und mit der Gruppe 9-Antiserum (1B), wurden unter 400-facher Vergrößerung betrachtet. Letzteres zeigt die &quot;Schwellung&quot; und Runden von Bakterienzellen in der Regel in einem positiven Quellung Reaktion gesehen. <img alt="Figur 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> 2. Zu schwer und zu leicht Inokula. Zubereitungen von der Pneumokokken-Zellsuspensionen, die zu schwer (2A) oder zu hell (2B) sind, wenn sie unter 400-facher Vergrößerung betrachtet.

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Die Quellung Reaktion ist der Gold-Standard-Technik für Serotypisierung Streptococcus pneumoniae. Diese Technik nutzt ein Mikroskop und Pneumokokken-spezifische Antiseren und wird häufig in Referenz-und Forschungslaboren weltweit eingesetzt.

Kapsel Serotypisierung von<em&gt; Streptococcus pneumoniae</em&gt; Verwenden der Quellung Reaktion

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

capsular-serotyping-streptococcus-pneumoniae-using-quellung

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K Aufrufe.  Murdoch Childrens Research Institute.  Die Quellung Reaktion ist der Gold-Standard-Technik für Serotypisierung Streptococcus pneumoniae. Diese Technik nutzt ein Mikroskop und Pneumokokken-spezifische Antiseren und wird häufig in Referenz-und Forschungslaboren weltweit eingesetzt. 

Serie: Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

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.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

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

Forschung

Immunologie und Infektion

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Live Cell Imaging von Bacillus subtilis Und Streptococcus pneumoniae Verwendung von Automated Zeitraffer-Mikroskopie

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

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.

Charakterisierung von Entzündungsreaktionen während der intranasalen Kolonisation mit Streptococcus pneumoniae

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.

Überwachen von Änderungen in Membran-Polarity, Membrane Integrity und intrazelluläre Ionenkonzentrationen in<em&gt; Streptococcus pneumoniae</em&gt; Verwenden von Fluoreszenzfarbstoffen

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 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.

Kapsel Serotypisierung<em&gt; Streptococcus pneumoniae</em&gt; Durch Latexagglutination

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

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Nicht-invasive Bildgebung von der angeborenen Immunantwort in einem Zebrafisch-Larven Modell<em&gt; Streptococcus iniae</em&gt; Infektions

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

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

A Murine Model of Group B Streptococcus Vaginal Colonization

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

Ein Mausmodell der Gruppe B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

A Robust Pneumonia Modell in intaktem Immunsystem Nagetiere zur Bewertung antibakterieller Wirksamkeit gegen<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; oder<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Intrakranielle Subarachnoidal Route der Infektion für die Untersuchung Rollen von Streptococcus Suis Biofilmen in Meningitis in einem Mausmodell der Infektion

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.