Das Projekt beschrieben wird durch Verleihungsnummer 5R01AI116895 aus dem Nationalen Institut für Allergien und Infektionskrankheiten unterstützt. Der Inhalt ist allein in der Verantwortung der Autoren und stellen nicht notwendigerweise die offizielle Position des Nationalen Instituts für Allergien und Infektionskrankheiten oder die National Institutes of Health vertreten.

1. Erstellen von Proben <ol><li> Wärme aktivieren C. difficile Sporen bei 65 ° C für 30 min und auf Eis lagern. Hinweis: C. difficile - Sporen hergestellt und gereinigt werden , wie zuvor beschrieben , 7,9,10. </li><li> Bereiten Sie die Keimung Lösung bei X + 1 ml, wobei X die Anzahl der Abtastwerte ist, die während des Assays entnommen werden. Anmerkung: Die Keimlösung ist spezifisch für die Spezies von Bakterien untersucht-Sporen werden. Für C. Testen difficile Sporenkeimung, verwendeten wir eine Lösung von 10 mM Tris (pH 7,5), 150 mM NaCl, 100 mM Glycin und 10 mM Taurocholsäure in deionisiertem Wasser. </li><li> Vor der Durchführung des Tests, ziehen eine 1,0 ml Probe der Keimungslösung ohne Sporen als Rohling für Cortex-Fragment Erkennung dienen. </li><li> Hinzufügen Sporen zu einer OD 600 von ~ 3,0 auf die Keimung Lösung und vortex schnell. Hinweis: Für unsere Keimung Studien in B. subtilis, wir haben das gleiche OD 600 Sporen als für C. difficile. Hinweis: Diese Menge an Sporen funktionierte gut für die Überwachung der Hirnrinde Fragment während C. difficile Spore Keimung. Die Menge von Sporen in diesem Assay verwendet wird, sollte experimentell für jedes Bakterium oder Belastung bestimmt werden. </li><li> Entfernen, um eine 1,2 ml Probe unmittelbar nach der Sporen Zugabe und Zentrifuge bei Raumtemperatur für 1 min bei 14.000 × g. Dies ist der Zeitpunkt Null. </li><li> Übertragen 1,0 ml des Überstands aus der zentrifugierten Probe in ein frisches Röhrchen zur anschließenden cortex Fragmentanalyse. Hinweis: Sollte DPA muss überwacht werden, eine separate 100 ul - Probe zum Nachweis von DPA in der Lösung zu entfernen unter Verwendung eines Assays auf Basis von Terbium - Fluoreszenz 9,18,19. </li><li> Fixieren Sie die gesammelte Probe bei -80 ° C. </li><li> Wiederholen Sie die Schritte 1.4 und 1.5 in regelmäßigen Abständen, bis alle Zeitpunkte abgeschlossen. Hinweis: Da der Zeitaufwand für die Zentrifugation und Probenentnahmen, Zeitpunkte weniger thein 2 min waren abgesehen nicht möglich während unseres Verfahrens, so dass unsere Abtastintervallen waren einmal alle 2 min für 14 min. </li><li> Als Positivkontrolle für Zucker Nachweis und die Quantifizierung Verringerung umfassen die folgenden Mengen an N Acetylglucosamin (nmol): 0, 12,5, 25, 50, 100, 250, 500 und 5.000. Bereiten Sie diese Proben in der gleichen Zeit wie Cortex Proben und führen zusammen mit den Cortex-Proben, so dass die Standard-Kurve an die Daten relevant sind, erzeugt durch Sporen keimen. </li><li> Nachdem alle Zeitpunkt Proben gesammelt werden, lyophilisieren die Proben. Anmerkung: Wir gefriergetrocknet, unsere Proben in 2 ml mit Schraubverschluss Röhrchen. </li></ol> 2. Messung Cortex Fragmente <ol><li> lyophilisierte Proben in 120 ul 3 N HCl supplementiert mit 1% Phenol und 0,5% β-Mercaptoethanol resuspendieren. </li><li> Übertragen Sie die resuspendierten Proben auf 2 ml mit Schraubverschluss Röhrchen. Hinweis: Für die Reproduzierbarkeit, sicherzustellen, dass alle die gefriergetrocknete Probe suspendiert und übertragen wird. </li> &lt;li&gt; Inkubieren der Proben in einem Umlaufwasserbad bei 95 ° C für 4 Stunden. </li><li> Nach der Inkubation, legen Sie die Proben auf Eis, bis sie kühl sind. </li><li> Neutralisieren der Proben mit 120 ul 3 M NaOH. </li><li> Hinzufügen, 80 ul einer gesättigten Natriumhydrogencarbonat-Lösung und 80 ul einer 5% Essigsäureanhydrid-Lösung zu jeder Probe und vortexen. </li><li> Inkubieren der Proben bei Raumtemperatur für 10 min. </li><li> Übertragen Sie die Proben zurück zu dem 95 ° C warmen Wasserbad für genau 3 min. Hinweis: Dieser Schritt ist zeitkritisch und längere Zeiten nachgeschaltete Signalentwicklung beeinflussen können. </li><li> Entfernen Proben aus dem Wasserbad und kühlen auf Eis. </li><li> In 400 ul 6,54% K 2 B 4 O 7 · 4H 2 O in jedes Röhrchen und Wirbel. </li><li> Inkubieren der Proben in einem Umlaufwasserbad bei 95 ° C für genau 7 min. Hinweis: Dieser Schritt ist zeitkritisch und längere Zeiten nachgeschaltete Signalentwicklung beeinflussen können. </li><li> Entfernendie Proben und auf Eis für 5 min. </li></ol> 3. Farbreagenz <ol><li> Während Proben auf Eis (Schritt 2.12) sind, bereiten Sie das Farbreagenz. Man löst 0,320 g p -dimethylaminobenzaldehyde (DMAB; Ehrlich-Reagens) in 1,9 ml Eisessig. Hinweis: Das Reagenz ist Zeit, Temperatur und lichtempfindlich und sollte nicht im Voraus gemacht werden. </li><li> Nachdem der DMAB vollständig auflöst, 100 ul 10 N HCl und Wirbel. </li><li> 5 ml Eisessig und Wirbel. </li></ol> 4. kolorimetrische Reaktion <ol><li> Transfer 100 ul jeder gekühlten Kortex Probe in ein neues 1,5 ml Mikrozentrifugenröhrchen. </li><li> In 700 ul der frisch zubereiteten Farblösung zu jeder Probe. </li><li> Unmittelbar übertragen Sie die Proben auf einem 37 ° C Wasserbad und für genau 20 min inkubiert. </li><li> Übertragen Sie 200 ul jeder Probe auf eine klare 96-Well-Platte. </li><li> Quantifizierung der Proben bei 585 nm auf einer PlatteLeser. Die Proben sollten bei einer gelben Farbe und eine positive Reaktion auf lila verschiebt starten. Je mehr Hirnrinde vorhanden ist, desto tiefer die violette Farbe. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+cortex+hydrolysis+precedes+DPA+release+during+Clostridium+difficile+spore+germination.">Spore cortex hydrolysis precedes DPA release during Clostridium difficile spore germination.</a> J Bacteriol. 197 (14), (2015).</li><li>Francis, M. B., Allen, C. A., Shrestha, R., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acid+recognition+by+the+Clostridium+difficile+Germinant+Receptor,+CspC,+is+important+for+establishing+infection.">Bile acid recognition by the Clostridium difficile Germinant Receptor, CspC, is important for establishing infection.</a> PLoS Pathog. 9, e1003356 (2013).</li><li>Moir, A., Smith, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genetics+of+bacterial+spore+germination.">The Genetics of bacterial spore germination.</a> Annu. Rev. Microbiol. 44, 531-553 (1990).</li><li>Zhang, P., Kong, L., Wang, G., Setlow, P., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Raman+tweezers+and+quantitative+differential+interference+contrast+microscopy+for+measurement+of+dynamics+and+heterogeneity+during+the+germination+of+individual+bacterial+spores.">Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores.</a> Journal of biomedical optics. 15, 056010 (2010).</li><li>Ghuysen, J. -. M., Tipper, D. J., Strominger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+that+degrade+bacterial+cell+walls.">Enzymes that degrade bacterial cell walls.</a> Methods Enzymol. 8, 685-699 (1966).</li><li>Park, J. T., Johnson, M. 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+submicrodetermination+of+glucose.">A submicrodetermination of glucose.</a> J Biol Chem. 181, 149-151 (1949).</li><li>Thompson, J. S., Shockman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+the+Park+and+Johnson+reducing+sugar+determination+suitable+for+the+assay+of+insoluble+materials:+its+application+to+bacterial+cell+walls.">A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls.</a> Anal Biochem. 22, 260-268 (1968).</li><li>Paredes-Sabja, D., Setlow, P., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SleC+is+essential+for+cortex+peptidoglycan+hydrolysis+during+germination+of+spores+of+the+pathogenic+bacterium+Clostridium+perfringens.">SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens.</a> J Bacteriol. 191, 2711-2720 (2009).</li><li>Paredes-Sabja, D., Sarker, M. 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Bacteriol. 192, 418-425 (2010).</li><li>Akoachere, M., 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=Indentification+of+an+in+vivo+inhibitor+of+Bacillus+anthracis+spore+germination.">Indentification of an in vivo inhibitor of Bacillus anthracis spore germination.</a> J. Biol. Chem. 282, 12112-12118 (2007).</li><li>Duncan, C. L., Strong, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+medium+for+sporulation+of+Clostridium+perfringens.">Improved medium for sporulation of Clostridium perfringens.</a> Appl Microbiol. 16, 82-89 (1968).</li><li>Heffron, J. D., Lambert, E. A., Sherry, N., Popham, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+anthracis+SleL+(YaaH)+protein+is+an+N-acetylglucosaminidase+involved+in+spore+cortex+depolymerization.">The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization.</a> J Bacteriol. 190, 7601-7607 (2008).</li><li>Wang, S., Shen, A., Setlow, P., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Dynamic+Germination+of+Individual+Clostridium+difficile+Spores+Using+Raman+Spectroscopy+and+Differential+Interference+Contrast+Microscopy.">Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy.</a> J Bacteriol. 197, 2361-2373 (2015).</li></ol>

The authors have nothing to disclose.

Nach Stimulation unterzogen Sporen den Prozess der Keimung verlieren ihre Beständigkeitseigenschaften ohne die Notwendigkeit einer makromolekularen Synthese. Wenn Sporenauskeimung ausgelöst wird, 2 die Spore Kern freigibt DPA im Austausch für Wasser. Aufgrund der hohen DPA - Gehalt der ruhenden Spore, die sporen Kern unter intensiver osmotischen Druck und dem Fach Peptidoglycan, Hirnrinde, hilft durch Einwirken vermutlich als Barriere für Expansions 2 den Kern aufquellen verhindern. <p clas...

Endospores metabolisch ruhenden Formen von Bakterien, die Bakterien erlauben, in ungünstigen Umgebungen zu bestehen. In vielen sporenbildenden Bakterien, wird die Sporenbildung von Nährstoffmangel induziert aber dieser Prozess kann 1 durch Änderungen der pH - Wert, der Exposition gegenüber Sauerstoff oder anderen Spannungen gesteuert werden. Während in ihrer metabolisch ruhenden Sporenform, wider Bakterien UV - Strahlung, Austrocknung, höhere Temperaturen und Einfrieren 2. Der größte Teil des Wissens über die Sporulation Prozess stammt aus Studien im Modellorganismus Bacillus subtilis. Die Sporulation Prozess beginnt mit der DNA - Replikation und die Bildung eines axialen Glühfaden 3,4. Eine asymmetrische Septum teilt sich dann die Zelle in zwei ungleich große Fächer. Die größere Kammer, die Mutterzelle, verschlingt die kleinere Kammer, die forespore. Sowohl die Mutterzelle und forespore Genexpression koordinieren, um die Sporen und die Mutterzelle zu reifen schließlich lysiert, die Freigabe der dormant Sporen in die Umgebung 1. Die Sporen Struktur und Zusammensetzung ist in vielen Bakterienarten konserviert. Die Spore Kern hat einen geringeren Wassergehalt als die vegetative Zelle und ist reich mit Dipicolinsäure (DPA) 1,2. Während die Sporenbildung wird DPA in die Spore Kern im Austausch für Wasser gepumpt. Den Kern umgibt , ist eine innere Membran sporen wo in den meisten sporenbildenden Bakterien, die Rezeptoren , die die kleinen Moleküle erkennen , die Keimung (Keimungsmittel) stimulieren 2 befindet. Das Hotel liegt etwas außerhalb der inneren Membran der Spore ist eine Schicht aus Zellwandpeptidoglycan. Eine spezialisierte Peptidoglycan Schicht (Cortex) umgibt die Peptidoglycan-Zellwand und viele der gleichen Komponenten wie Zellwandpeptidoglycan [alternierende N-Acetylglucosamin (NAG) und N-Acetylmuraminsäure (NAM)] zusammengesetzt ist. Jedoch etwa 50% der NAM - Reste in der Hirnrinde haben muramic-δ-lactam 5,6 umgewandelt </sup&gt;. Während der Sporenkeimung, diese muramic-δ-Lactam-Reste werden durch die Sporenrinde lytische Enzyme (SCLEs) erkannt, so dass die Rinde ermöglicht abgebaut werden (ein Prozess wesentlich Keimung abzuschließen), aber nicht die Zellwand. Die Rinde umgibt , ist eine äußere Membran und mehrere Schichten von Hüllproteinen 2. den Prozess der Keimung Steuerung ist von entscheidender Bedeutung für die sporenbildende Bakterien. Die Keimung wird eingeleitet , wenn Keimungsinduktor Rezeptoren interagieren mit ihren jeweiligen Keimungsmittel 2. In vielen sporenbildende Bakterien sind diese Keimungsmittel Aminosäuren, Zucker, Nukleotide, Ionen oder Kombinationen davon . 2 C. difficile Sporenkeimung durch Kombinationen bestimmter Gallensäuren, [zB Taurocholsäure (TA)] und Aminosäuren (beispielsweise Glycin) 7-10 eingeleitet. Obwohl es Unterschiede zwischen der C. difficile Keimung Weg und die untersuchten Wege in andere sporenbildendeBakterien, wie B. subtilis, allen gemeinsam ist die absolute Notwendigkeit der Spore Kortex abzubauen eine vegetative Zelle zu ermöglichen , von der gekeimten Spore 2,8 zu wachsen. Cortex Abbau kann durch die SCLEs CwlJ / SLEB erreicht werden oder SLEC (wie in B. subtilis gefunden) (wie in vielen Clostridia gefunden). Cortex Hydrolyse verringert die Beschränkung der Zellwand und der Spore. Dies ermöglicht eine volle Kern Rehydratisierung notwendiger Schritt in 2 viele der Proteine ​​, die für den Zellstoffwechsel zu reaktivieren. Wenn Sporen keimen, die ruhende Sporen ändert sich von einem phasen hellen Zustand in einen Phase-Dunkel-Zustand und dieser Prozess kann durch eine Veränderung der optischen Dichte (OD) bei 600 zu messen nm 11. Ein früherer Bericht legt nahe , dass ein großer Teil dieser Änderung in der OD auf die Freisetzung von DPA aus der Spore 12 zurückzuführen ist. Während unserer Studie haben wir versucht , den Zeitpunkt C zu vergleichen difficile Sporenkeimung und überwacht dieFreisetzung von DPA und Cortex - Fragmente 9. Für diese Studie war es wichtig, die Freisetzung von Cortex-Fragmente zu überwachen, wie sie begann von keimen Sporen freigesetzt werden. Die kolorimetrischen Test hier verwendet wurde , basiert auf einem Verfahren zur Bereitstellung von Zuckern mit reduzierenden Enden entwickelt Ghuysen et al erfasst wird . 13. Weil andere 14 - Protokolle zum Nachweis von reduzierenden Zuckern beschrieben haben oder diese Protokolle 15 modifiziert, kann die Literatur zu diesem Thema verwirrend sein. Hier stellen wir ausführlich die Schritt- für -Schritt - Verfahren zur kolorimetrischen Detektion von Zuckern befreit keimen C. Reduktions difficile - Sporen. Obwohl diese Studie verwendet C. difficile - Sporen sind die reduzierenden Zucker während der Keimung von Sporen von anderen sporenbildenden Bakterien freigesetzt können mit diesem Protokoll 9,16,17 erkannt werden.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Tabelle 1 zeigt typische Ergebnisse mit NAG - Standards. Daten verwendet werden , um eine Standardkurve zu erzeugen. Die Tabelle 2 zeigt typische Ergebnisse von einem Keimungsassay in Keimung Puffer , ergänzt mit 100 mM Glycin und 10 mM TA (Keimung fördernden Bedingungen) oder 100 mM Glycin allein (nicht-keimende Bedingungen). In Abwesenheit von TA, C. difficile - Sporen keimen nicht , und es gibt wenig Änderung in Gegenwart von cortex Fragme...

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Erkennen Cortex Fragmente während der bakteriellen Sporenauskeimung

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Research

JoVE Journal

Immunology and Infection

9.3K Aufrufe.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Serie: Detecting Cortex Fragments During Bacterial Spore Germination

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Propagierung und Erkennen einer ansteckenden Molecular Clone von Maedi-Visna Virus, das Green Fluorescent Protein exprimiert

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Forschung

Immunologie und Infektion

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Elektroporation von Functional bakterielle Effektoren in Säugerzellen

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Auswertung der extrazellulären Vesikel Funktion im Malaria-Infektion

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Methoden zur Erkennung von zytotoxischen amyloiden folgende Infektion der pulmonalen Endothelzellen durch Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

Spore-Adsorption als ein Nonrecombinant Display-System für Enzyme und Antigene

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Ein High-Throughput- Shigella-spezifischen bakterizide Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...