Bu çalışma Bill ve Melinda Gates Vakfı (PneuCarriage proje, Grant 52099) ve Victoria Hükümeti&#39;nin Operasyonel Altyapı Destek Programı tarafından desteklenmiştir. Eileen Dunne, Janet Strachan, ve yazının eleştirel okuma için Kim Hare teşekkürler.

1.. Yazma Hücre Süspansiyon hazırlanması <ol><li> Steril tek kullanımlık 1 ul döngü kullanarak, katı seçici olmayan at kanlı agarda pnömokokların taze bir gecede saf kültür küçük süpürme almak ve 100 ul Kalp İnfüzyon (HI) suyu aşılamak. Süspansiyon, sadece gözle görülür bulanık olarak görünmelidir. Emülsiyon haline getirmek için hafifçe ajite tüp. Not: Brain Heart Infusion suyu gibi diğer ortam da uygun olabilir. Serumu (% 10, örneğin bir nihai konsantrasyon (h / h) at serumu) ve kullanılmadan önce yaklaşık olarak 1 saat boyunca 37 ° C&#39;de inkübasyon eklenmesi etkisini artırmak için kullanılabilir. </li></ol> 2. Hücre Süspansiyon Yoğunluğu denetleme Adım 1.1 &#39;de hazırlanan hücre süspansiyonu da negatif kontrol olarak kullanılacak, ve yazma sera eklemeden önce, hücre yoğunluğu için kontrol edilmelidir. <ol><li> 1 ul döngü kullanılarak, glas üzerine aşama 1.1 &#39;de hazırlanan aşı bir damla aktarmaks mikroskop lamı. </li><li> Bir çift forseps kullanarak, süspansiyon üzerinde bir küçük dairesel lamel yerleştirin. </li><li> Faz-kontrast ayarı kullanılarak, süspansiyonun yoğunluğu (Şekil 1A) kontrol etmek için mikroskopik 400X büyütme altında hazırlanmasını dikkate alınmalıdır. <ol><li> Aşılama (Şekil 2A) çok ağır ise, seyreltilmesi aşama 1.1 &#39;de yapılan hücre süspansiyonu daha fazla suyu ekleyin. Tekrar hafifçe karıştırın. </li><li> Aşılama çok açık ise, örneğin, hücreler, 50 aşamasında 1.1 &#39;de yapılan hücre süspansiyonu hazırlamak için daha fazla bakteri ekleyin ve yavaşça karıştırın, mikroskobik alan (Şekil 2B) görünür &lt;bulunmaktadır. Not: deneyimi ile, hücre süspansiyonu yoğunluğu sallayarak ve sadece gözle görülür bulanık görünmelidir ışığa kadar tutarak karar olabilir. </li></ol></li><li> Hücrelerin uygun bir sayısı mikroskopik alanına görebilir kadar yineleyin 2,1-2,3 adımları tekrarlayın. Not: Cam slaytlar ve coverslips kavuz tehlikesi. Kullanımdan sonra, slaytlar ve lamelleri enfeksiyöz materyal ile kontamine olan ve biyolojik tehlikeli kesici kabın içine atılmalıdır. </li></ol> 3. Yazma <ol><li> Cam mikroskop lamı üzerine (örneğin, 1 ul döngü kullanılarak) hazırlanmış inokulum ~ 1 ul aktarın. Biohazard kesici kabın içine döngü atın. </li><li> Slayt üzerine oda sıcaklığı antiserumu eşit bir hacmi (1 ul halka dolusu) ilave edilir ve iki döngü ile iyi damla karıştırın. </li><li> Bir çift forseps kullanarak, damla dokunmamaya özen, süspansiyon üzerinde küçük dairesel lamel yerleştirin. Kurumasını slaytta sıvı önlemek için hemen antiserumların ilave edildikten sonra süspansiyon lamel yerleştirin. </li><li> Bir mikroskop kullanarak, 400X büyütme ile faz-kontrast altında süspansiyon gözlemlemek. Pnömokok hücre &#39;şişme&#39; ya da çok az 40 ° C&#39;de görülebilir zaman negatif bir kapsül şişme deneyi reaksiyonu görülmektedirHiçbir antisera ile pnömokokal hücre süspansiyonu olarak gözlemlenen ile benzer 0X büyütme, (negatif kontrol) ilave edildi. Negatif kontrol hücreleri ile karşılaştırıldığında hücre büyütülmüş ya da &quot;şişmiş&quot; ve daha görünür olduğunda A pozitif bir kapsül şişme deneyi reaksiyon gözlenmektedir. Not: Şüpheli reaksiyonlar, oda sıcaklığında 5-10 dakika inkübasyondan sonra, ya da daha fazla antiserum ile reaksiyonu tekrarlanarak reaksiyonun incelenmesi, örneğin daha araştırılmalıdır. </li><li> Bir pozitif reaksiyon görülmektedir kadar bireysel antiserumları havuzları ardışık test ile pnömokok Serotipleme Davranış. Alternatif olarak, havuz serotiplerinin yerel sıklığı bağlı olarak &#39;tur&#39; de uygulanabilir. Not: &#39;mermi&#39; Ölçme en yaygın serotiplerine antikorları içeren havuzlar ilk test edilmesini sağlar, çaba ve maliyet en aza indirir. Birkaç havuzları Eşzamanlı test de interp yararlı olabilir, hangi ek negatif kontrolleri sağlarretation. Belirli bir &#39;yuvarlak&#39; tüm havuzları negatif ise, havuzları sonraki mermi ile izolat test edin. </li><li> Reaktif havuzun içinde bulunan uygun türü veya grup antiserum kullanarak izolat test edin. Uygun olduğunda, daha fazla serotip ayırt etmek için etken antisera kullanın. Not: Üretici tarafından verilen pnömokok antiserumlara Keys uygun havuz, tip, grup veya faktör antiseranın ve (herhangi bir çapraz reaksiyonlar dahil) sonuçlarının yorumlanması seçiminde kullanılmaktadır. </li><li> Olumsuz sonuçları tüm havuzları ile elde edilirse, (91 serotiplere 21 karşı antikorlar içerir) Omniserum reaktif ile izolat test edin. Omniserum reaktif ile izole bir pnömokok test ederken olumlu bir kapsül şişme deneyi reaksiyon hala görülmez ise izolatın az veya hiç kapsül ifade veya serotip karşı antikorlar Omniserum temsil olmadığını, çünkü bu olabilir. İkinci kategori, yeni serotiplerini içerebilir. </li> &lt;/ Ol&gt;

<ol>
	<li>O'Brien, K. L., 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=Burden+of+disease+caused+by+Streptococcus+pneumoniae+in+children+younger+than+5+years:+global+estimates.">Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.</a> Lancet. 374, 893-902 (2009).</li><li>Bogaert, D., De Groot, R., Hermans, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+colonisation:+the+key+to+pneumococcal+disease.">Streptococcus pneumoniae colonisation: the key to pneumococcal disease.</a> Lancet Infect. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+pneumococcal+serogroups+cause+the+most+invasive+disease:+implications+for+conjugate+vaccine+formulation+and+use,+part+I.">Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I.</a> Clin. Infect. Dis. 30, 100-121 (2000).</li><li>Johnson, H. L., 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=Systematic+evaluation+of+serotypes+causing+invasive+pneumococcal+disease+among+children+under+five:+the+pneumococcal+global+serotype+project.">Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five: the pneumococcal global serotype project.</a> PLoS Med. 8, (2010).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378, 1962-1973 (2011).</li><li>Obaro, S. K., Adegbola, R. A., Banya, W. A., Greenwood, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carriage+of+pneumococci+after+pneumococcal+vaccination.">Carriage of pneumococci after pneumococcal vaccination.</a> Lancet. 348, 271-272 (1996).</li><li>Mulholland, K., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+after+pneumococcal+vaccination.">Serotype replacement after pneumococcal vaccination.</a> Lancet. 379, 1388-1389 (2012).</li><li>Austrian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quellung+reaction,+a+neglected+microbiologic+technique.">The quellung reaction, a neglected microbiologic technique.</a> Mt. Sinai. J. Med. 43, 699-709 (1976).</li><li>Konradsen, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+serotyping+of+Streptococcus+pneumoniae+in+Europe.">Validation of serotyping of Streptococcus pneumoniae in Europe.</a> Vaccine. 23, 1368-1373 (2005).</li><li>Lovgren, 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=Evolution+of+an+international+external+quality+assurance+model+to+support+laboratory+investigation+of+Streptococcus+pneumoniae,+developed+for+the+SIREVA+project+in+Latin+America,+from.">Evolution of an international external quality assurance model to support laboratory investigation of Streptococcus pneumoniae, developed for the SIREVA project in Latin America, from.</a> J. Clin. Microbiol. 45, 3184-3190 (2007).</li><li>Reasonover, A., 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=The+International+Circumpolar+Surveillance+interlaboratory+quality+control+program+for+Streptococcus+pneumoniae.">The International Circumpolar Surveillance interlaboratory quality control program for Streptococcus pneumoniae.</a> J. Clin. Microbiol. 49, 138-143 (2011).</li><li>Hare, K. 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=#34;Dodgy+6As&amp;#34;:+differentiating+pneumococcal+serotype+6C+from+6A+by+use+of+the+Quellung+reaction.">#34;Dodgy 6As&amp;#34;: differentiating pneumococcal serotype 6C from 6A by use of the Quellung reaction.</a> J. Clin. Microbiol. 47, 1981-1982 (2009).</li><li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+cpsA-cpsB+sequence+polymorphisms+and+serotype-/group-specific+PCR+to+predict+51+Streptococcus+pneumoniae+capsular+serotypes.">Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes.</a> J. Med. Microbiol. 52, 1047-1058 (2003).</li><li>Castillo, D., 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=Identification+and+characterization+of+Streptococcus+pneumoniae.">Identification and characterization of Streptococcus pneumoniae.</a> Laboratory Methods for the Diagnosis of Meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. 2nd ed. , 73-86 (2011).</li><li>Kaijalainen, T., Rintamaki, S., Herva, E., Leinonen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene-technological+and+conventional+methods+in+the+identification+of+Streptococcus+pneumoniae.">Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae.</a> J. Microbiol. Methods. 51, 111-118 (2002).</li><li>Sorensen, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typing+of+pneumococci+by+using+12+pooled+antisera.">Typing of pneumococci by using 12 pooled antisera.</a> J. Clin. Microbiol. 31, 2097-2100 (1993).</li><li>Diagnostica SSI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+to+pneumococcal+factor+antisera.">Key to pneumococcal factor antisera.</a> , (2013).</li><li>Diagnostica SSI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumococcal+antisera+for+in+vitro+diagnostic+use.">Pneumococcal antisera for in vitro diagnostic use.</a> , (2011).</li><li>Lund, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+diagnosis+of+Pneumococcus+infections.">Laboratory diagnosis of Pneumococcus infections.</a> Bull. World Health Organ. 23, 5-13 (1960).</li><li>Satzke, C., 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=Standard+method+for+detecting+upper+respiratory+carriage+of+Streptococcus+pneumoniae:+Updated+recommendations+from+the+World+Health+Organization+Pneumococcal+Carriage+Working+Group.">Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group.</a> Vaccine. 32, 165-179 (2013).</li><li>Vernet, G., 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=Laboratory-based+diagnosis+of+pneumococcal+pneumonia:+state+of+the+art+and+unmet+needs.">Laboratory-based diagnosis of pneumococcal pneumonia: state of the art and unmet needs.</a> Clin. Microbiol. Infect. 17, 1-13 (2011).</li><li>Casewell, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiences+in+the+use+of+commercial+antisera+for+the+capsular+typing+of+klebsiella+species.">Experiences in the use of commercial antisera for the capsular typing of klebsiella species.</a> J. Clin. Pathol. 25, 734-737 (1972).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu teknikte kritik bir safha, saf kültür çorbasına bakteriyel hücrelerin uygun bir sayısı ile birlikte ekleme süspansiyonunu hazırlamak için kullanılır sağlamaktır. Bu agar plakaları üzerinde büyüme ve koloni morfolojisi, farklı pnömokok izolatı arasında değişmekte olduğu not edilmelidir. Bazı döngü ile kapalı almak için ve suyu iyi emülsifiye olmayan zor olabilir, kuru ve sert koloniler üretir. Hücre süspansiyonunun hazırlanması, bu nedenle, bakterilerin daha büyük bir tarama gerekebilir. Her durumda, bu göreceli boyutu antisera olan hücreler ile karşılaştırıldığında, böylece tek tek hücreler, ve agregalar, negatif kontrol olarak ilave ortada şarttır. Buna ek olarak, çok sayıda bakteri hücreleri kapsül antijeninin fazla kapsül şişme deneyi reaksiyonu 10 inhibe edebilir bir yanlış negatif tepkimeye neden olabilir. Bu nedenle, uygun bir hücre yoğunluğuna sahip bir bakteriyel süspansiyon (Şekil 1) oluşturmak için önemlidir. Suspansiyon konsantre edilmiş, formalin birkaç damla eklenmesi ile birkaç ay boyunca muhafaza edilebilir. Bu kolay toplu testini sağlar ve aynı zamanda laboratuvar kaynaklı enfeksiyonların riskini azaltarak suyu sterilize, bu zehirli, aşındırıcı, kanserojen, duyarlılaştırılmaları ve yanıcı taşıma formalin güvenlik riskine karşı tartılır olmalıdır rağmen. Buna ek olarak, bazı izolatlar formalin (veriler gösterilmemiştir) mevcudiyetinde tepkileri zayıf olduğunu gözlemledik. Kapsül şişme deneyi reaksiyonları bir mikroskop kullanılarak incelenir. Faz kontrast kullanılması, kapsülün görünürlüğünü artırır. Bazı laboratuarlarda, reaksiyon 1,000 X büyütme 14, 16 petrol-batırma kullanılarak izlenebilir. Buna ek olarak, bazı laboratuar ayrıca bir pozitif reaksiyon 10, 16, 22 görünürlüğünü artırmak için bir karşı leke (metilen mavi) kullanın. Burada sunulan yöntem, yağ-i olmaksızın görülüyormmersion ve 400X büyütme 15, 17 altında. Bu yöntem, hızlı, basit ve 23, okumak için nispeten kolaydır. Bu antiserumlar ve test prosedürleri sırayla sağlamak için iyi bir iç kalite kontrol prosedürleri korumak için önemlidir. Bir dış kalite güvence programına katılım genellikle faydalıdır. Pnömokok olarak serolojik tipleme için kapsül şişme deneyi reaksiyonun önemli bir sınırlama gerekli olan antiserumların yüksek maliyetidir. Bununla birlikte, bu durum, her bir reaksiyon için kullanılan antiserumun küçük bir miktarına gerek yukarıdaki protokol en aza indirilir. Diğer bazı yöntemler pnömokok 23, 24 serotip için tarif edilmiştir. Ancak, kapsül şişme deneyi altın standart olmaya devam etmektedir ve yeni yöntemlerin geliştirilmesi kapsül şişme deneyi reaksiyonu 23 karşı standardizasyonu gerektirir. Uygun anti-serumlar kullanılarak, reaksiyon, kapsül şişme deneyi başka kapaklar belirlemek ve yazmak için kullanılabilirule bakteri 25 üretir. Kapsül şişme deneyi reaksiyon mevcut ve gelişmekte olan pnömokok serotipleri karakterizasyonu, yanı sıra aşı etkinliği ve taşıma ve hastalık üzerine aşılamanın uzun vadeli etkileri hakkında bilgi veren vesile oluyor.

Streptococcus pneumoniae (pnömokok) hastalık yükü kaynak yoksul ülkelerde 1 ağırlıklı olarak düşen, yılda beş yaşın altında yaklaşık 800.000 çocuk ölümünden sorumlu. Pnömokok hastalıkları örneğin sepsis, menenjit ve zatürre 2 gibi ciddi hayatı tehdit eden enfeksiyonlar, akut otitis media gibi lokalize enfeksiyonlar şiddeti değişir. Pnömokok 90 üzerinde kapsüler serotip 3, 4, tarif edilmiştir. Mevcut pnömokok aşıları invasif hastalık 5, 6 çoğunluğu için sorumlu olan serotip karşı koruma sağlar. Bununla birlikte, aşıların yaygın kullanımı, nazofarenks taşıyıcı ve invasif hastalık 7, 8, hem nonvaccine serotiplerinin aşıda bulunan serotiplerin değiştirilmesi ile ilişkilendirilmiştir. Bu epidemiyolojik için serotipleme önemini vurgulamaktadırgözetim ve uzun vadeli aşı etkisi 9 inceler. Pnömokok tiplemesi için altın standart yöntem, 1902 yılında ilk Neufeld tarafından açıklanan kapsül şişme deneyi reaksiyonu olarak bilinen kapsül reaksiyon testi, (Avusturya 10 bakınız). Uyum yüksek derecede laboratuarlar arasında ve kapsül şişme deneyi reaksiyon ve diğer serotipleme yöntemler 11-13 arasında bulunmuştur. Kapsül şişme deneyi reaksiyonu 10, 14-16 çeşitli varyasyonları vardır. Burada tarif edilen metot, bir karşı leke veya yağ daldırma objektif kullanımını gerektirmeyen, daha ziyade, hazırlanan numune bir damla yazarak serumu ile karıştırılır ve 400X büyütme altında hemen incelenmiştir. Reaksiyon genellikle kapsül şişme deneyi ticari olarak temin edilebilen anti-serumlar kullanılarak gerçekleştirilir. Burada tarif edilen yöntem, daha düşük maliyetli yapmak, bazı başka yöntemler 16, 17 oranla daha az anti-serumlar kullanır. Bir kez isolate bir pozitif reaksiyon gözlenmiştir kadar sekans antisera havuzları ile test edilir, bir pnömokok olarak tanımlanır. Her havuz antiserum 91 pnömokok serotipleri 18 karşı oluşturulan antiserumların farklı karışımları içerir. Havuz kurulduktan sonra, reaktif havuzda içerdiği bireysel tip antisera ve grup sırayla ayrı ayrı test edilmiştir. Grubu antisera (örneğin, Grup 22 antiserum serotip 22F ve 22A ile reaksiyona girer), belirli bir grup içindeki tüm serotipler ile reaksiyona iken, tip antisera, genel olarak, tek bir serotip (örneğin Tip 1 antiserum, serotip 1 ile reaksiyona giren) ile reaksiyona girer. Gruplar içindeki bazı serotip başka bir faktör, antisera (örneğin, serotip 22F faktör 22b ile reaksiyona girer, ancak 22c) kullanılarak ayrılır. Serotip 19, 20, tespit edilene kadar, bu durumda test ilgili tüm etken antisera ile devam edilir. Reaksiyon, kapsül şişme deneyi basit ve 14 hızlı ve herhangi bir l yapılabiliraboratory bu iyi kaliteli mikroskop antiserumlar ve uygun bir dizi ile donatılmıştır.

<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

Streptococcus pneumoniae kapsül 90 farklı serotip (pnömokok) vardır. Yanı sıra pnömokok epidemiyolojisi anlamak için bir araçtır olarak, kapsül serotipleme aşı etkinliği ve darbe çalışmaları için yararlı bilgiler sağlayabilir. Kapsül şişme deneyi Reaksiyon pnömokok kapsüler serotiplemeye altın standart yöntemdir. Bir yöntem olup, kapsüler polisakarite karşı havuzlanmış ve spesifik antiserumlarla bir pnömokok kökenli hücre süspansiyonu test içerir. Antijen-antikor reaksiyonları mikroskopik olarak gözlenir. Bir bakteri hücre süspansiyonu 1) hazırlanması, bir cam slayt üzerinde hücrelerin ve antiserumlar 2) karıştırma, ve 3) bir mikroskop kullanılarak kapsül şişme deneyi tepki okuma: Protokol üç ana adım vardır. Reaksiyon, kapsül şişme deneyi gerçekleştirmek için oldukça basit ve uygun bir mikroskop ve antisera mevcuttur yerde uygulanabilir.

<html> Tip-spesifik antikorun, kırılma endeksi 10 bir değişime yol açan, pnömokokun kapsüle bağlar olumlu bir kapsül şişme deneyi reaksiyon meydana gelir. Mikroskop altında bakıldığında, bakteriler &quot;şişmiş&quot; ve 14 daha görünür. Şekil 1A HI suyu içinde pnömokok hücrelerini göstermektedir. Tip-spesifik anti-serum bakteriyel süspansiyona ilave edildiğinde, pnömokokki (Şekil 1B), &quot;şişmiş&quot; kırılma ve daha yuvarlak görünür. Brota ilave çok fazla bakteri hücreleri hücre toplama ya da yanlış negatif bir reaksiyona neden olabilir. (Şekil 2A) ya da &quot;çok hafif &#39;(Şekil 2B)&#39; de ağır&quot; in HI suyu içinde pnömokok kökenli hücrelerin uygulanması gibi 2: Şekil. 08fig1highres.jpg &quot;src =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; Şekil 1. Negatif ve pozitif kapsül şişme deneyi reaksiyon. Serotip 9V pnömokok hazırlıklar hiçbir antiserum (1A) ve grup 9 antiserum ile izole (1B), 400X büyütme altında incelendi. Bu ikinci &quot;şişme&quot; ve genellikle bir pozitif kapsül şişme deneyi reaksiyonunda görülen bakteriyel hücrelerin yuvarlama göstermektedir. <img alt="Şekil 2," fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> Şekil 2. Çok ağır ve çok hafif inokula. 400X büyütme altında incelendi (2A) veya çok hafif (2B) çok ağır pnömokok hücre süspansiyonları, preparatlar.

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

Kapsül şişme deneyi Reaksiyon Streptococcus pneumoniae serotip için altın standart bir tekniktir. Bu teknik mikroskop ve belirli pnömokok antiseranın kullanır ve yaygın dünya çapında bir referans ve araştırma laboratuvarlarında kullanılır.

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-streptococcus-pneumoniae-using-quellung

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K Görüntüleme.  Murdoch Childrens Research Institute.  Kapsül şişme deneyi Reaksiyon Streptococcus pneumoniae serotip için altın standart bir tekniktir. Bu teknik mikroskop ve belirli pnömokok antiseranın kullanır ve yaygın dünya çapında bir referans ve araştırma laboratuvarlarında kullanılır. 

Video: 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.

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

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.

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 ...

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 ...

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 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 ...

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.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

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.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

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.

Immünkompetan Kemirgenler bir Sağlam Pnömoni Modeli karşı antibakteriyel etkinliğini değerlendirmektir<i&gt; S. pneumoniae</i&gt;<i&gt; H. influenza</i&gt;<i&gt; K. pneumoniae</i&gt;<em&gt; P. aeruginosa</em&gt; ya da<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.

Kafa içi Subarachnoidal rota için soruşturma rolleri streptokok suis biyofilmler menenjit bir fare enfeksiyon modeli içinde enfeksiyon

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.