Çalışmalar AdvanDx tarafından desteklenen edildi.

1. Örnek Toplama ve Hazırlama <ol><li> Sepsis şüphelenilen hastalarda venöz kan toplayın ve bir kan kültürü seti birlikte oluşan iki BACTEC (BD, Sparks, MD) kan kültürü şişeleri, bir aerobik ve anaerobik bir koymak. </li><li> BACTEC 9240 araç içine yerleştirin şişeler 35 ° C </li><li> Enstrüman kan kültürü şişesi organizmaların büyüme nedeniyle alarm tetiklenir kadar inkübe edin. </li><li> Kan kültürü aracı şişe çıkarın. </li></ol> 2. Boyama için önce Reaktiflerin Hazırlanması <ol><li> 4 ml, 240 ml taze deiyonize veya distile su Boyama Bulaşık doğrudan takip 60x Yıkama Çözeltisi ekleyerek yıkama solüsyonu hazırlayın. </li><li> Isıtmak için 55 ° C su banyosunda boyama kabına koyun. </li><li> 2-8 kalan konsantre ° C depolayın </li><li> Montaj Orta buzdolabından çıkartın ve oda sıcaklığında. </li></ol> 3. Gram Boyama <ol><li> Yavaşça girdap kan kültürü şişesi. </li><li> , Bir iğne ve şırınga ya da bir alt kültüre aparatları kullanarak, kan (yaklaşık 5 ml), steril vida kapaklı tüp içinde şişe ve yerden kaldırmak. </li><li> Steril bir pipet kullanarak, büyük bir damla kan bir cam mikroskop lamı üzerine yerleştirin. </li><li> Hava slayt kuru ve 10 saniye boyunca metanol düzeltmek. </li><li> Hava kuru kaydırın İzin. </li><li> Gram kan kültürü şişesi büyüyen organizmanın morfolojisini belirlemek için kan filmi leke gerçekleştirin. </li><li> Immersiyon yağı lens kullanarak, slayt görünümünde. Çiftleri ve Gram boyama gözlenen kısa zincirleri Gram pozitif koklar enterokok türleri ile en tutarlı. </li><li> Bu Gram boyama sonucu bakteriyel kimlik kullanmak için uygun PNA BALIK prob kiti seçimi sürücüler. Bu kan kültürü E. kullanılarak boyandı. faecalis / OE PNA BALIK leke. </li></ol> 4. Lekelere PNA BALIK <ol><li> Tüp kan içeren smear hazırlama önce dönen hafifçe karıştırın. </li><li> Fiksasyon Çözüm PNA BALIK Mikroskop Slayt iyi bir damla yerleştirin. </li><li> 10 mcL pozitif kan kültürü ile tüp ya da küçük bir damla Fixation Çözüm transferi ve emülsifiye hafifçe karıştırın. </li><li> 20 dakika ısıtarak smear sabitleyin. 55 ° C ısıtma plakası. </li></ol> 5. Kalite Kontrol Malzeme Bir pozitif ve bir negatif kalite kontrol slayt boyama slaytlar her parti ile test edilmelidir. <ol><li> E. kullanın. faecalis / OE Kontrol, bu amaç için AdvanDx satın alınan ya da E. referans suşları sıvı kültürlerden gelen smear hazırlamak Slaytlar faecalis ve E. faecium olarak ayrı slaytlar veya bir slayt ve Negatif Kontrol malzemesi olarak Staphylococcus spp Streptococcus spp karışık ya Pozitif Kontrol. </li></ol> PNA metodoloji hücre duvarı penetrasyon optimize etmek için tasarlanmış olduğundan QC sonuçları, uygun test koşullarında, özellikle hibridizasyon darlığı ve hücre duvarı penetrasyon etkileyen izlemek için gerekir 6. Melezleme <ol><li> E. bir damla ekleyin faecalis / OE PNA smear ile mikroskop lamı üzerine iyi. </li><li> Lamel ekleyin. Hava kabarcıkları kaçının. </li><li> 30 inkübe ± 5 dk. 55 ± 1 ° C ısıtma plakası </li><li> Slayt taşıyıcıya inkübasyon yer slaytlar ardından </li></ol> 7. Sıkı Yıkama <ol><li> Önceden ısıtılmış Yıkama Çözüm daldırın slayt taşıyıcı 55 ° C ve lamel dikkatlice çıkarın. Genellikle, lamel nazikçe yıkayın Çözüm slayt çalkalayarak slaytlar. Bazen, lamel forseps ile yavaşça itti olmalıdır. </li><li> 30 yıkama çözüm ± 5 dakika inkübe edin. 55 ± 1 ° C </li><li> Yıkama çözüm slaytları çıkarın </li><li> Hava kuru slayt izin ver </li></ol> 8. Montaj <ol><li> Her bir slayt için bir damla Montaj Orta ekle </li><li> Lamel ekleyin. Hava kabarcıkları kaçının. </li><li> 2 saat içinde bir floresan mikroskop, slayt inceleyin. </li><li> Slaytları floresan soğutulması için yol olarak güneş ışığı veya diğer güçlü ışık kaynaklarına doğrudan maruz bırakmayın. </li></ol> 9 - Sonuçların Yorumlanması Slayt inceleme için kullanılan floresan mikroskop AdvanDx Dual Band Filtresi ve 60x veya 100x petrol amacı ile donatılmış olmalıdır. QC slaytlar bu hibridizasyon meydana geldi onaylamak için ilk incelenmiş olmalıdır E. faecalis bakış birden fazla alanlarda parlak yeşil floresan koklar gibi görünmelidir ve E. faecium parlak kırmızı koklar gibi görünecektir . Sigara enterokok kontrol slayt nonfluorescent görünmelidir. Kontroller sistem onayladıktan sonra, hasta slaytlar incelenebilir. İlk kan film kırmızımsı görünür ama parlak kırmızı ve yeşil koklar oldukça belirgin olacaktır. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Şekil 1" /> Yeşil-pozitif E. Şekil 1 Temsilcisi örnekler faecalis (solda), kırmızı pozitif E. faecium (orta) ve negatif (sağ) test sonuçları. 10 Temsilcisi Sonuçlar Üç kurum, bu PNA BALIK leke değerlendirilmesi ve gözden geçirilmiştir kısaltılmış 1.5 saat protokol 2.5 saat protokol karşılaştıran bir çok merkezli bir klinik araştırmaya dahil edildi. 152 rutin Gram pozitif koklar çiftleri ve zincirleri olmak üzere toplam (GPC) pozitif kan kültürü şişeleri çalışmalar dahil edildi. % 100 (152/152) modifiye sonuçları arasındaki anlaşma ve E. için orijinal deneyi prosedürü vardı faecalis / OE PNA BALIK: 41/41 E. faecalis; 33/33 Diğer enterokoklar ve 78/78 Diğer GPC (Tablo 1). Bu boyama yöntemi bu klinik çalışma sırasında (Tablo 2) enfes bir duyarlılık ve özgüllük vardı. <table border="1"><tbody><tr><td></td><td> Rutin Yöntemleri </td><td> E. faecalis / OE PNA BALIK Prosedürü </td><td> E. faecalis / OE PNA BALIK Kısa Prosedürü </td></tr><tr><td> E. faecalis </td><td> 41 </td><td> 41 (Yeşil Pozitif) </td><td> 41 (Yeşil Pozitif) </td></tr><tr><td> E. faecium </td><td> 27 </td><td> 27 (Pozitif Kırmızı) </td><td> 27 (Pozitif Kırmızı) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (Pozitif Kırmızı) </td><td> 2 (Pozitif Kırmızı) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (Pozitif Kırmızı) </td><td> 2 (Pozitif Kırmızı) </td></tr><tr><td> Diğer Enterococcus spp. </td><td> 2 </td><td> 2 (Pozitif Kırmızı) </td><td> 2 (Pozitif Kırmızı) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 (Negatif) </td><td> 17 (Negatif) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (Negatif) </td><td> 33 (Negatif) </td></tr><tr><td> S. mitis </td><td> 1 </td><td> 1 (Negatif) </td><td> 1 (Negatif) </td></tr><tr><td> S. pyogenes </td><td> 3 </td><td> 3 (Negatif) </td><td> 3 (Negatif) </td></tr><tr><td> S. bovis </td><td> 2 </td><td> 2 (Negatif) </td><td> 2 (Negatif) </td></tr><tr><td> S. salivarius </td><td> 1 </td><td> 1 (Negatif) </td><td> 1 (Negatif) </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2 (Negatif) </td><td> 2 (Negatif) </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7 (Negatif) </td><td> 7 (Negatif) </td></tr><tr><td> Diğer Streptococcus spp. </td><td> 7 </td><td> 7 (Negatif) </td><td> 7 (Negatif) </td></tr><tr><td> Abiotrophia spp. </td><td> 3 </td><td> 3 (Negatif) </td><td> 3 (Negatif) </td></tr><tr><td> Peptostrepococcus spp. </td><td> 2 </td><td> 2 (Negatif) </td><td> 2 (Negatif) </td></tr><tr><td> Toplam </td><td> 152 </td><td> 152/152 100% Anlaşması </td><td> 152/152 100% Anlaşması </td></tr></tbody></table> Tablo 1. bakteri Listeleme standart (2.5hr) ve hızlı (1,5 saat) protokolü kullanılarak test edilmiştir. <table border="1"><tbody><tr><td> Hassasiyet E.faecalis </td><td> Hassasiyet Diğer Enterococcus spp. </td><td> Özgüllük </td></tr><tr><td> % 100 (41/41) % 95 CI (93,0-100) </td><td> % 100 (33/33) 33 / 33 % 95 CI (91,3-100) </td><td> % 100 (78/78) % 95 CI (96,2-100) </td></tr></tbody></table> Tablo 2 - 1.5 saat PNA BALIK protokol duyarlılığı ve özgüllüğü .

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <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+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

Bu video-makale üretim AdvanDx sponsor oldu. Benjamin Kristal, bu makalede kullanılan araçlar ve reaktifler yapar AdvanDx, bir çalışanın.

E. enterococcus faecalis / OE PNA FISH testinin standart kültür yöntemleri daha 2-3 gün önce kimlik sağlayabilir. Tayini için kısaltılmış bir prosedür (1.5 hourr), antimikrobiyal tedavi ve hasta sonucu daha iyi bir yaklaşım için kullanılabilecek hızlı bir şekilde tespit sağlar. Bunu desteklemek için bir çalışmada Forrest ve ark. (6) tarafından yapıldı. 2005 yılından itibaren birbirini takip eden iki yıl içinde mikrobiyoloji laboratuvarına konvansiyonel mikrobiyolojik yöntemlerle kan kültürü şişeleri büyüyen ve 2006 yılında E. ekleyerek çiftleri ve zincirleri Gram pozitif koklar tespit faecalis / OE PNA BALIK. Buna ek olarak kurumları antimikrobiyal ekibi (AMT) tarafından geliştirilen bir tedavi algoritması zamanında laboratuvar tarafından oluşturulan PNA BALIK verileri etkin bir şekilde kullanmak. Değerlendirilir Primer sonuç, kan kültürü zaman önce etkili antimikrobiyal tedavi uygulanması çizmek ve PNA BALIK laboratuarlar iş akışı içine tesis sonra oldu. Hastalık, hasta konumu ve ampirik antimikrobiyal tedavi şiddeti ölçüldü. Bakteriyemi enterokok elde edilen 224 hasta hastane ile toplam 129 ve 95 PNA BALIK dönemde müdahale öncesi dönemde değerlendirildi. PNA BALIK E. tespit geleneksel kültürlerin (1.1 vs 4.1 gün, p &lt;0.001) daha önce faecalis 3 gün. PNA BALIK medyan 2.3 gün önce E. faecium tespit (1.1 vs 3.4 gün, p &lt;0.001) ve etkili bir tedavi (1.3 ve 3.1 gün, p &lt;0.001) başlatarak için zaman içinde istatistiksel olarak anlamlı bir azalma ile ilişkili ve 30 gün azaldı mortalite (% 26 vs% 45, p = 0.04). Bu grup sonucuna E. AMT bir tedavi algoritması ile birlikte faecalis / OE PNA FISH testinin elde edilen hastane ile bakteriyemi E. faecium hastalar için uygun ampirik antimikrobiyal tedavinin erken başlamasına yol açmıştır . <ol><li> Etiketleri ve reaktifler üzerinde yazılı olan son kullanma tarihleri, herhangi bir şekilde değiştirilmiş olmamalıdır sonra Reaktifler kullanılmamalıdır. Bunlar kullanılmadığı zaman buzdolabında kalmalıdır veya onlar potens kaybedebilirsiniz. </li><li> Kan kültürü şişeleri örnekleme önce karıştırılması gerekmektedir. </li><li> Bu malzeme slaytlar arasındaki çapraz kontaminasyon neden, ya da reaktif kontaminasyona neden olabilir çünkü kan yaymasında damlalıklı şişe ucu dokunmak için izin vermemesi için önemlidir. </li><li> Su banyosu ve ısıtma bloğu sabit bir 55 ° C&#39;de muhafaza edilir emin olun. </li><li> Dual Band Filtresi veya renkler açıkça ayırt olmayacaktır dışında filtre kullanmayın. </li><li> Mikroskop Slaytlar veya hibridizasyon olmayacağına dışında mikroskop lamı kullanmayın. </li><li> Mikroskop düzgün çalıştığını önemlidir. Mikroskop ampul kullanımı için saat onaylanmış sayısı içinde olduğundan emin olun. </li></ol><ul><li> Bazı nadir meydana gelen Enterococcus türleri, E. asinii, E.dispar, E. haemoperoxidus, E. cecorum, E. columbae, E. saccharolyticus, E. solitariusa ve E gibi diğer Enterococcus türleri, literatürde PNA probu tarafından tespit edilmez . kükürtlü. </li><li> E. moraviensis E. olarak tanımlanır dizisi benzerlikleri nedeniyle faecalis. </li><li> Streptococcus anginosus bazı suşları dizisi benzerlikleri nedeniyle, sahte bir yeşil olumlu floresan üretir . </li><li> VersaTREK kan kültürü şişeleri klinik çalışmalar sırasında değerlendirilir değildi. E. algılamak için performans faecalis ve diğer Enterococcus spp. VersaTREK kan kültürü şişeleri ile bilinmemektedir. </li><li> Yanlış pozitif yeşil otofloresans standart bir FITC filtresi Dual Band Filtresi yerine kullanılması durumunda ortaya çıkabilir. </li><li> Yanlış negatif sonuçlar nadiren karışık büyüme nedeniyle veya tahlil tekniği hata nedeniyle oluşabilir. </li><li> Kullanılan enstrümantasyon tipine ve durumuna, elde edilen görüntünün görünümünü etkiler. Floresan kullanılan mikroskop, ışık kaynağı ve hücrelerde rRNA düzeyi varydue olabilir </li><li> Katı ortam izolasyonu, diğer organizmalar ile karışık bir büyüme ayırt etmek ve negatif sonuç veren pozitif kan kültürleri tanımlamak için ihtiyaç vardır. </li><li> Ürün, kan kültürlerinden başka örnekler ile geçerli olmamıştır. </li></ul>

Reaktifler E. faecalis / OE PNA BALIK aşağıdaki kit bileşenleri oluşmaktadır: <ul><li> Fiksasyon Çözüm Fixation Çözüm deterjan ile tuzlu fosfat tamponlu 3 ml. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 1.5 mL PNA hibridizasyon probları. çözüm. 30% formamid içerir. </li><li> Deterjan ile 60x Yıkama Çözüm 60x Yıkama Çözeltisi 50 ml Tris tamponlu salin </li><li> Orta gliserol olarak Orta 3 mL photobleaching inhibitörü Montaj Montaj </li><li> Kalite kontrol slaytları </li><li> <a href="http://www.advandx.com">AdvanDX</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Enterokoklar E. ile bakteriyemi yaygın bir nedenidir. faecalis, E. faecium baskın tür . Çünkü E. ampisilin ve vankomisine direnç faecalis, E. faecium direnci hala yaygın karşılaştırıldığında, enterokok türler arasındaki farklılaşmayı sağlayan hızlı testlerin geliştirilmesi, uygun tedavi ve direnç gözetim için önemlidir. E. faecalis OE PNA FISH testinin (AdvanDx, Woburn, MA) in situ hibridizasyon formatında türe özgü bir floresan peptid nükleik asit (PNA) problar kullanır ve 1,5 saat ve türe özgü önemli bilgiler veren potansiyel sonuçlarını bir süre sunuyor tedavi. Çok Merkezli 1.5 saatlik E. performansını değerlendirmek için çalışmalar yapıldı faecalis / OE PNA BALIK prosedürü orijinal 2,5 saat metodlara ve pozitif kan kültürü şişesi doğrudan enterokokların belirlenmesi için standart bakteriyoloji yöntemlerine göre.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

In situ hibridizasyon tayininde floresan bir Peptid Nükleik Asit (PNA BALIK) kullanarak bir pozitif kan kültürü, Enterococcus faecalis ve diğer Enterococcus türleri doğrudan tanımlanması için hızlı bir protokoldür.

Belirlenmesi için 1.5 Saat Usulü Enterococcus</emDoğrudan Kan Kültürleri> Türler

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.8K Görüntüleme.  Cedars-Sinai Medical Cente. In situ hibridizasyon tayininde floresan bir Peptid Nükleik Asit (PNA BALIK) kullanarak bir pozitif kan kültürü, Enterococcus faecalis ve diğer Enterococcus türleri doğrudan tanımlanması için hızlı bir protokoldür.

Video: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Kompleman tükenmesi Özel Hücre Populasyonlarının tükenmesi

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

İmmünoloji ve Enfeksiyon

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Kan örneklerinden Bakteriyel Patojen Algılama ve Antimikrobiyal Direnç Testi için bir günlük iş akışı şeması

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

İlköğretim İnsan Keratinositler gelen Organotipik Radye Kültürlerin Üretimi

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Kordon Kanı gelen Prekürsör B-hücreli altkümeleri İzolasyonu

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Toplam Protein Ekstraksiyon ve 2-D Jel Elektroforez Yöntemleri<em&gt; Burkholderia</em&gt; Türler

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Bir Kuş Türlerinin Sindirim Sistemi geçtikten sonra Bulaşıcı prions belirlenmesi için prosedürler

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF kütle spektrometrisi kullanılarak kan kültür besi yerinden, Gram negatif bakterilerin hızlı belirlenmesi

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Hızlı Bakteriyel Teşhis ve Antibiyotik Duyarlılık Testi için bir Kan Kültürü Peleti hazırlanması

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Belirlenmesi için 1.5 Saat Usulü Enterococcus Türler