<html> &quot;Pozitif&quot; olarak 1. Kan Kültürü et suları Bayrağı <ol><li> Sürekli izleme kuluçka kabine işaret kan kültürü şişesini çıkarın ve biyolojik güvenlik kabini içine yerleştirin. Not: Şişeler tehlikeli mikroorganizmaları ihtiva edebilir ve evrensel önlemleri takip edilmesi gerekir. Nedeniyle örnekleme bulaşıcı aerosollerin riskinden dolayı, tüm numune alma işlemleri, bir Biyogüvenliği Sınıf II düzgün akış kabininde gerçekleştirilmelidir. </li></ol> 2.. Gram Leke Hazırlanan olduğunu <ol><li> Yerel kurumsal protokollere uygun olarak bildirilir kan kültürü sıvısından bir Gram boya hazırlayın. Not: Gram negatif organizmalar mikroskobu ile tespit edildiğinde kan kültür brotu aşağıdaki yöntemi ile işlenir. Gram pozitif organizmalar tespit edilirse, gen belirlenmesini ve direnç işaretçileri hedef alternatif bir moleküler metodu (bu makalede açıklanan) suyu 13 uygulanır. </li></ol>Bir Serum Ayırma Tüp Bayraklı Kan Kültürü Broth&#39;u e &quot;&gt; 3. Transferi <ol><li> Yavaşça 2-3x ters çevrilerek kan kültürü şişesi karıştırın. </li><li> Biyogüvenlik kabini içinde bir emniyet kan transfer cihazı için 10 ml şırınga takın. </li><li> Kan kültürü şişesine kan transfer cihazını takın ve enjektörün içine et suyu 5 ml çekilme. Bir serum ayırma tüpüne aspire BC suyu aktarın. </li></ol> Kan Kültürü Broth 4. Konsantrasyon <ol><li> Kırmızı kan hücrelerinin büyük bir ses çıkarır, 15 dakika boyunca 1250 x g&#39;de santrifüj tüpü serum ayırma. </li><li> Aspire ve hemen jel / sıvı arayüzü üzerindeki ince beyaz tabakada yaklaşık 1 ml bırakmak için dikkatli olmak, steril bir transfer pipet kullanarak süpernatant atın. Not: aerobik şişe jel / sıvı arayüzü opak beyaz renkli görünen ile tüpün dibine kırmızı kan hücreleri net bir ayrım göstermektedir. Buna karşılık, zamanJel / sıvı arayüzü olan lize alyuvar hücresi bileşenleri süpernatan içinde süspansiyon haline getirilmiş, kalan bir koyu kırmızı bir renk olarak görünür litik anaerobik kan kültürü suyu uygulanır. </li></ol> 5.. Santrifugasyon Yıkama Adımları Tekrarla <ol><li> Yavaşça steril bir pipet ile jel arayüzü üzerinde beyaz tabaka sıvının son 1 ml karıştırın ve daha sonra 1.5 ml mikrosantrifüj tüp içine tüm hacim transferi. 30 saniye boyunca 288 x g &#39;de yeni tüp santrifüjleyin. </li><li> Yeni bir 1.5 ml mikrosantrifüj tüpü içine plastik bir tek 1 ml transfer pipeti kullanarak süpernatant aktarın ve pelet ile tüp atın. Not: Bu, bakım pelet transferini engellemek için alınır, bu aşamada çok önemlidir. Bu yüzer madde pigmentli kalır ve topak her zaman açık bir şekilde görülmez gibi anaerobik BC şişeden işlenen bir numune için özellikle önemlidir. </li></ol> Kalıntı Hücreler 6. Lysis </p&gt; <ol><li> Santrifüj 1 dakika sonra aspiratı (ve artıklar) için 18,407 xg&#39;de örnek pelet bozmadan mümkün olduğunca az kalan sıvıyı terk ince uçlu bir transfer pipet kullanarak yüzer. </li><li> Aşağı yukarı pipetleme ve steril DNase ve RNase içermeyen su 1 ml kalıntı pelletini. Not: Bazı yazarlar, canlı olmayan bakteri hale pelletini steril su yerine alkol çözeltilerin kullanımını tarif etmişlerdir. </li><li> 1 dakika boyunca 18.407 x g&#39;de yeniden süspansiyon haline çözeltisi santrifüjleyin. </li></ol> Bakteriyel Proteinlerin 7. Ekstraksiyon <ol><li> Aspire ve yine mümkün olduğu kadar sıvı kaldırılır sağlamak ince bir ucu pipet ile aspirasyon kullanarak, süpernatant atın. </li><li> 10 ul formik asit (% 70 v / v) içindeki pelet tekrar. Not: deri ile temas ederse o solunması veya eğer Karınca asidi vücudu etkileyebilir. Solüsyonları hazırlamak veya manipüle zaman rpersonel kişisel koruyucu donanımlar için ek bir duman kabine kullanmak ecommended. </li><li> Bir homojen yeniden süspanse çözüm sağlamak için pipet kullanarak iyice karıştırın. Fiziksel olarak bozmak için pelet pipet kullanarak, daha homojen bir çözelti sağlamak için gerekli olabilir. </li></ol> MALDI-TOF 8. Hazırlanması Hedef Plaka <ol><li> Hedef nokta başına hazırlanan süspansiyonun 2 ul temiz bir MALDI-TOF hedef plaka inoküle. Başarısız okur zaman sorunun üstesinden gelmek için her örnek için 3 hedef siteleri hazırlayın. </li><li> MALDI-TOF hedef plakası kurumasını bekleyin. Kurutma hızı plaka üzerinde hava akımı üst düzeye çıkarmak için, davlumbaz kenarında plaka yerleştirilerek artırılabilir. </li><li> Matris solüsyon içerisinde 1 ul (10 mg / ml α-siyano-4-hidroksisinamik asit (HCCA),% 50 asetonitril,% 2.5 trifloroasetik asit) ile, her bir nokta Bindirme kurutuldu. Not: Bu conta girdiği eğer solunması veya eğer Matrix çözümü vücudu etkileyebilirderi ile ct. Hazırlamak veya çözümler manipüle zaman personel, kişisel koruyucu donanımlar için ek bir duman kabine kullanmanız önerilir. </li><li> MALDI-TOF hedef plakası yukarıda tarif edildiği gibi çeker ocak kenarında kurumaya bırakın. Homojen bir hazırlık plaka üzerinde hedef noktalardan her doldurur emin olun. </li></ol> Kütle Spektrometre içine 9. Sıra MALDI-Hedef Plaka <ol><li> MALDI-TOF kütle spektrometresi içine MALDI-TOF hedef plakası yerleştirin. Plaka yaylı plakaları ile aynı hizada oturuyor olun. Not: Tüm hedef noktalar kuru kadar hedef plakası takmayın. </li><li> Kauçuk conta gerekli vakum şartlar oluşturulabilir izin tiftik, toz ve saç temiz olduğundan emin olun. </li><li> MALDI-TOF sahne kapağını kapatın ve daha sonra cihazın önündeki &quot;/ out&quot; düğmesine basın. Kapak şimdi kilitlemek ve gerekli vakum yaratılmış olmasını sağlayacaktır. </li></ol> 10. MALDI-TOF MS Spectra Acquisition <ol><li> MALDI Biyotiplendirme sonucunda ve FlexControl yazılımını açın. </li><li> Yazma yazılım içinde &quot;dosya&quot; başlıklı açılır menüden &quot;yeni sınıflandırma&quot; seçeneğini seçin. Projeyi adlandırın ve sonra &quot;yeni&quot; seçeneğini seçin. Yazma yazılım içinde kurulumunu tamamlamak için &quot;İleri&quot; yi seçin. </li><li> Kontrol yazılım penceresinde hedef plaka ekrandaki grafik tanımlamak. Farenin sol tuşuna basılı tutarak aşılanmış noktalar üzerinde fareyi hareket ettirerek kullanımda MALDI-TOF hedef noktalar vurgulayın. </li><li> Seçilen hedef konum üzerinde herhangi bir yere tıklayın ve ardından açılır menüden &quot;analitlerle eklemek&quot; seçmek için sağ fare düğmesini kullanın. </li><li> Hedef noktalar her biri için &quot;Kimlik&quot; sütununa kan kültürü, kimlik bilgileri ekleme ve &quot;next&quot; Tamamlandığında tıklayın. </li><li> Ticari &quot;taksonomi&quot; spektrumları veritabanı seçin ve &quot;next&quot; seçeneğini seçin. Not: Labolaboratuarları referans spektrumları sayısını artırmak için içi veya ticari veritabanları ek kullanabilir. </li><li> &quot;Bitirmek&quot; tıklayın ve MALDI-TOF MS spektrumları üreten başlayacaktır. Not: MALDI-TOF ayarları fabrika ayarları (lineer pozitif mod, 60 Hz lazer frekansı, 20 kV hızlandırma voltajı, 16.7 kV IS2 gerilimi, 170 NSEC çıkarma gecikmesi ve 2,000-20,137 m / z aralığı) başına idi. </li><li> MALDI kontrol yazılımı tepe oluşturmak için başarısız olursa, spektrumları elle elde etme (yöntem tarif edilmemektedir) ile gerçekleştirilebilir. </li></ol> MALDI-TOF MS Puanlarının 11. Mesaj Analizi Yorumlama <ol><li> Spektrumları edinilen ve analiz tamamlandıktan sonra, her hedef için kimlik listesini genişletmek için Yazma yazılım türleri kimlik yanındaki &quot;+&quot; düğmesini seçin. </li><li> En iyi 5 benzer eğer belirterek, ilk 5 tanımlamaları çalışma. Skorları aynı hedefin ≥ 1.7 ile uyumsuz ilk 5 cinsSpot karışık Brotların olasılığını yükseltti. Not: MALDI-TOF teknoloji karışık Brotların saptanması için düşük bir duyarlılığa sahiptir. Bir karma suyu türler içerir, örneğin, yüksek bir güven puanı karışık türlerin sadece biri için tespit edilebilir. </li><li> Bir puan ≥ 2.0, bir hedef noktada en yüksek maç için dikkat edildiğinde türler rapor. </li><li> Hedef noktada en yüksek puanı 1.7 ≥, ancak &lt;2.0, sadece cins rapor zaman. Ilk 5 teşhislerin ek kriterler uyumlu iseniz, o tür düzeyinde rapor. </li></ol> MALDI-TOF Hedef Plate 12. Kaldırma <ol><li> Tüm spektrumları MALDI-TOF makinede &quot;out / in&quot; düğmesine tam basın zaman. </li><li> MALDI-TOF hedef plakası yuvasını açın ve MALDI-TOF hedef plakasını çıkarın. </li><li> Bir yeniden MALDI-TOF hedef plakası, alkol kullanılarak temiz tüm hedef noktalar kullanarak ve temiz ve kuru bir kez oda sıcaklığında MALDI-TOF plaka saklayın. </li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Santrifüj sonrası adımlar ayrılmış eser bileşenler için değil, yeterli bakım ile gerçekleştirilen bir kan kültürü suyu için MALDI-TOF MS uygularken önemlidir. Bu, MALDI-TOF spektrumları ile müdahale sivri üretebilir hemoglobin da dahil olmak üzere kan kültürü bileşenleri ve insan hücresel proteinleri, kaldırmak için özellikle önemlidir. MS türler için ≥ 2.0 puan kesti ve cins tanımlanması için ≥ 1.7 tavsiye üretmektedir rağmen, diğer raporlar M.Ö. ?...

Nedeniyle bakterilere kan dolaşımı enfeksiyonu (BSI) olan hastalar 6-48% 1 arasında değişen, yüksek hastane içi mortalite devam etmektedir. Uygun ampirik antibiyotik teslim hayatta teşvik ve ağır sepsis hastalarının alt kümesi, uygun tedaviye her saat gecikme sağkalım 2,3 azalmış ilişkilidir. Buna göre, klinik laboratuvar önemli bir hedefi hızla saptamak, tanımlamak ve klinik kararlarını bildirmek için kan kültürlerinde bakteri varlığını iletişim kurmaktır. Bu mikrobiyoloji laboratuvar son zamanlarda Gram leke 4 raporlanması ve zamanında antimikrobiyal tedavinin üzerinde en büyük etkiye sahip olduğu kanıtlanmıştır, gözlemsel bir çalışma göstermiştir ki, uçuş kütle spektrometresi matris destekli lazer desorpsiyon / iyonizasyon süresi (MALDI-TOF MS) kan kültür sıvılarında doğrudan uygulanan Gram negatif bakterilerin 5 neden BSI üçte biri reçeteleme etkileyebilir. 6,7 mikroorganizmaların tespit edilmesi için etkili bir laboratuar aracı yol açmıştır. Teknoloji artık iyi kurulmuş ve katı ortamlar 6,8 izole mikroorganizmaların hızlı ve doğru tanımlanması için pek çok laboratuvarda entegre edilmiştir. Kan kültürü çünkü düşük maliyetle mikroorganizmaların önceki bir kimlik elde etmek için potansiyeli hem klinisyen ve laboratuvar yöneticileri için mikroorganizmalar temyiz için &quot;olumlu&quot; sinyalini (BC) suyu MALDI-TOF MS doğrudan uygulama. Gram negatif bakteriler arasında değişen başarıyla tespit raporları ile kimlik standart fenotipik kültürü bazlı yöntemler ile karşılaştırıldığında kan kültürü suyu için MALDI-TOF MS doğrudan uygulama klinik yarar görülmüştür hassasiyetleri geniş ile sınırlı olmuştur 47-98,9 % 9-11. Duyarlığındaki farklılaşmaya muhtemelBC suyu bileşim, başlangıç ​​bakteri konsantrasyonu, numune hazırlama yöntemi değişkenlik hem de çalışma popülasyonları 9 karşılaşılan Gram negatif organizmaların dizisine ilişkindir. Bu yayınlanmış protokoller ile karşılaştırıldığında Burada yer alan yöntem, etanol, amonyum klorür ya da ek (non-matrix) asetonitril kullanımını önler. Bunun bir sonucu olarak, bakteri peleti potansiyel fenotipik duyarlılık testi yöntemleri suyu içinde, bu organizmalar için direkt olarak uygulanabilir için izin (protein ekstraksiyon noktaya kadar) uygun bir kalır. Buna ek olarak, sunulan yöntem, pahalı olmayan, güvenilir ve süre 12 &#39;eller&#39; minimal ile kan kültürü Gram leke sonuç 25 dakika içinde mevcut bakteriyel tanımlama ile hızlı olduğu gösterilmiştir. Bu yöntem, Gram negatif b tanımlamak için pozitif kan kültür sıvılarında doğrudan uygulanan formik asit çıkarma kullanan basit bir in-house spin-parçalama protokoldürMALDI-TOF MS teknolojisi ile Ancak bakteriler.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

Klinik mikrobiyoloji laboratuvarında önemli bir rol kan dolaşımı enfeksiyonu neden bakterilerin hızlı bir şekilde tanınmasını sağlamaktır. Geleneksel kimlik sadece olgunlaşmış katı agar kolonileri sonra geçerli bir kimlik belgesi ile sinyalize kan kültür sıvısının alt-kültürü gerektiriyor. MALDI-TOF MS katı ortam üzerinde koloniler uygulanan klinik olarak ilgili bakterilerin çoğunluğunun belirlenmesi için güvenilir ve hızlı bir yöntemdir. Bu bakteri türlerinin belirlenmesi hızlandırmak ve klinik yönetimini geliştirmek için bir potansiyele sahip olarak doğrudan kan kültür çorbasına MALDI-TOF MS uygulaması çekici bir yaklaşımdır. Bununla birlikte, üstesinden gelmek için önemli bir sorun kaldırılmaz ise, MS spektrumları müdahale ve yetersiz ya da düşük ayrımcılık kimlik puan neden olabilir, kan kültürü örneklerinde içerdiği müdahale reçineler, protein ve hemoglobin ön analiz çıkarılmasıdır. Buna ek olarak Bacter konsantre gereklidiria yeterli kalitede spektrumları geliştirmektir. Sunulan yöntem olup, konsantrasyon, arıtma ve bir işaret kan kültürü sıvısından bakterilerin erken tespiti için izin Gram negatif bakterilerin çıkarılması açıklanmaktadır.

Oluşturulan MALDI-TOF MS spektrumları spektrumları entegre referans veri tabanı ile karşılaştırılmaktadır. Bir logaritmik puan olası cins düzeyine kimlik (Şekil 1) ve türlerinin olası tanımlanması için ≥ 2.0 (Şekil için gerekli bir skor ≥ 1.7 tavsiyesi ile test izolatı ve referans veritabanı izolatları arasındaki maçın güven için atanır 2). Yazma yazılımı ile eşleşen skoru ≥ 1.7 ve ilk 5 tanımları (Şekil 1) <su...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

Uçuşu (MALDI-TOF) kütle spektrometresi (MS), doğrudan kan kültür çorbasına matris destekli lazer desorpsiyon / iyonizasyon zaman uygulanması bakteri tanımlanmasını hızlandırır. Sunulan yöntemi doğrudan kan kültür besi yerinden, Gram negatif bakterilerin tanımlanması için hızlı ve güvenilir bir yöntemdir.

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 ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K Görüntüleme.  Westmead Hospital.  Uçuşu (MALDI-TOF) kütle spektrometresi (MS), doğrudan kan kültür çorbasına matris destekli lazer desorpsiyon / iyonizasyon zaman uygulanması bakteri tanımlanmasını hızlandırır. Sunulan yöntemi doğrudan kan kültür besi yerinden, Gram negatif bakterilerin tanımlanması için hızlı ve güvenilir bir yöntemdir. 

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

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

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

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

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

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 ...

İmmünoloji ve Enfeksiyon

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Sıcak Sulu-fenol Ekstraksiyon tarafından Gram-negatif bakteriler arınma ve Lipopolisakkaritlerinin Görselleştirmesi

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Flourogenik DNAzymes kullanarak Bakterilerin Tespiti

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Tüm hücre MALDI-TOF Kütle Spektrometre Makrofaj Aktivasyon Farklı Modu analiz için Doğru ve Hızlı Yöntem

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Böbrek gelen Çift Negatif αβ T Hücreleri İzolasyon

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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 ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavansin için Başvuru sıvı mikrodilüsyon yöntemi<em&gt; İn Vitro</emAerobik Olarak Büyüyen Bakterilerin&gt; Duyarlılık Testi

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA gen Sıralama ve MALDI-TOF MS tarafından Nadir Bakteriyel patojenlerin tanımlanması

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Makrofaj koloni stimüle edici faktör farklılaştırılmış insan monosit türevli makrofajlar Kültür

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...