Laboratuvarda Biyofilm ilgili çalışmaları hibe tarafından finanse Diş ve Kraniofasial Araştırma ve Ulusal Alerji ve Bulaşıcı Hastalıklar (JLL-R). Enstitüsü Ulusal Enstitüsü R21DE017294 ve R21AI080930 sayılı. Içeriği sadece yazarların sorumluluğundadır ve mutlaka NIDCR NIAID veya NIH resmi görüşlerini temsil etmemektedir.

1. C. hazırlanması albicans C. albicans Risk Grubu 1/BSL1 mikroorganizma. Her zaman bu mikroorganizma ile çalışmak için iyi aseptik / steril teknikleri kullanmak ve biyolojik zararlı maddeleri uygun şekilde bertaraf edilmesi için kurumsal prosedürleri takip etmek hatırlıyorum. <ol><li> C. bir gecede kültür hazırlayın C tek bir koloni aşılanarak YPD (Maya Peptonlu Dekstroz) sıvı ortamda albicans YPD 25 ml içine albicans. </li><li> 30 ° C gecede orbital çalkalayıcı (yaklaşık 180 rpm) kültür inkübe edin. En C. albicans suşlarının, bu koşullar altında maya hücreleri gibi büyüyecek. </li><li> Santrifüj gecede kültürleri (yaklaşık 3000 rpm, 5-10 dakika), steril PBS ile iki kez hücreleri yıkayın ve RPMI 1640 ortamın pH 7.0 ve önceden ısıtılmış 165 mM morpholinepropanesulfonic asit ile tampon 20-25 ml nihai pelet tekrar süspansiyon 37 ° C (bu ortamda artık sadece &quot;RPMI 1640&quot; olarak anılacaktır). </li><li> Hemasitometre kullanarak hücreleri saymak. Sayımından sonra, 1.0 x 10 6 hücre / mL RPMI 1640 son bir yoğunlukta hücre süspansiyon hazırlamak. Not: hücrelerinin toplu bir eğilim olduğundan, yıkamalar arasında ve pipetleme önce şiddetle vorteks önemlidir. </li></ol> 2. Biyofilm Oluşumu için 96-iyi microtiter Plaka Ayarlama <ol><li> Bir çok kanallı pipet kullanarak C. 100 mcL ekleyin 96-iyi mikrotiter plate (-s) seçilen kuyulara albicans süspansiyon. Bu negatif kontrol olarak hizmet verecek, sütun 12 kuyu hücreleri eklemek etmeyin. </li><li> Kapak orijinal kapağı ile tüm mikrotiter plaka, mühür parafilm ile, 37, 24 saat için bir kuluçka ve inkübe içinde yer ° C. Inkübasyon süresi belirli deneysel tasarım adapte edilebilir. - 48 saat (yani 2, 4, 8, 12, 24 ve 48 saat) farklı zaman noktalarında birden fazla plakalar ekim ve her plaka işleme Örneğin, (24) bir süre üzerinde biyofilm oluşumu kinetiği incelemek mümkündür </li><li> Biyofilm oluşumundan sonra, çok kanallı pipet kullanarak dikkatle kuyuların her birinde oluşan biyofilm gibi dokunmatik ve bozmak için değil orta aspirat. </li><li> Bir çok kanallı pipet yıkama plakaları steril PBS (ortalama 200 mcL) üç kez kullanılması. Alternatif olarak, otomatik bir mikrotiter tabak yıkama kullanın. Yıkar yılları arasında, özellikle son yıkamadan sonra, herhangi bir kalıntı PBS kaldırmak için kağıt havlu ile blot ters bir pozisyonda plakaları boşaltın. Bu noktada kuyu alt kısmında oluşan biyofilm çıplak gözle bile açıkça görülebilir olmalı ve aynı zamanda ters bir mikroskop kullanılarak (Şekil 1) görüntülenebilmekte. Biyofilmler antifungal duyarlılık testi testleri için işlenmek üzere hazır. (Deneyin temel amacı, biyofilm oluşumu ölçüde değerlendirmek için ise, plakaları kolorimetrik yöntemi kullanılarak işlenecek hazır. XTT / menodion reaktifi (bkz. Adım 3) Bunun için ortaya çıkan renk olabilir ) mikrotiter plate okuyucusu kullanılarak okunabilir. </li></ol> 3. Biyofilmler Antifungal duyarlılık testleri <ol><li> Stok solüsyonları veya toz, RPMI 1640 ortamında test edilecek her antifungal çalışan bir solüsyon hazırlanır. Tipik yüksek konsantrasyonu 1.024 mg / ml flukonazol için ve 16 mcg / ml amfoterisin B ve kaspofungin. Diğer konsantrasyonları farklı ajanlar için kullanılıyor olabilir. </li><li> Bir çok kanallı pipet kullanarak, mantar biyofilm içeren her mikrotiter plate 1. sütununda karşılık gelen kuyulara antifungal yüksek çalışma konsantrasyonu 200 mcL ekleyin. </li><li> 2 - 10 sütunlar, her kuyuya RPMI 1640 100 mcL ekleyin. </li><li> Sütun 11 kuyulara RPMI 1640 100 mcL ekleyin. </li><li> Kolon 1 kuyulardan antifungal ajan 100 mcL çıkarın ve sütun 2 bitişik kuyuları (zaten 100 mcL orta içeren) ekleyin. </li><li> Iyi bir seri katlanan seyreltme gerçekleştirmek için yukarı ve aşağı pipetleme içeriğini karıştırın ve ipuçları kaldırmak. </li><li> Karıştırıldıktan sonra 10 sütun kuyulardan son 100 mcL hacmi atılır sonra sütun 10 kuyu, kadar doğru hareket tekrarlayın. Bu şekilde, bir dizi ilgi ajan (-ler) dilüsyonları iki katına, 10 sütun, kuyu en konsantre sütun 1 kuyu en çok konsantre gelen. 11 sütun rakipsiz biyofilm pozitif kontrol olarak görev yapacak ve 12 sütun boş kuyular negatif kontrol olarak görev yapacak. </li><li> 37 kapaklar, 24 -48 saat parafilm ve inkübe mühür ile plakalar Kapak ° C </li><li> Kuluçka döneminden sonra (3 x PBS) önce Adım 2.4 &#39;gibi plakalar yıkayın. </li><li> Bir çok kanallı pipet kullanarak arka plan X ölçümü için negatif kontrol kuyuları için iyi bir ön yıkanmış biyofilm içeren her yanı sıra 100 mcL XTT / menodion çözüm eklemekTT-kolorimetrik düzeyleri. <ol><li> XTT 0.5 g / L, 0.22 mikron gözenek boyutu filtre kullanarak filtre ile sterilize edilmiş olması gerekir steril Ringer laktat, PBS veya tuzlu doymuş bir çözüm olarak hazırlanmıştır. XTT çözümü ışığa karşı hassastır, bu yüzden hazırlanması sırasında alüminyum folyo ile kaplanmış olmalıdır. -70 Hazırlandıktan sonra ve filtre ile sterilize edilmiş 10 mL çalışma birimlere kısım, ve mağaza ° C Alüminyum folyo kullanarak ışık tüpleri koruyun. Kullanımdan hemen önce belirli bir deney için gerekli olarak birçok boru türü olarak sadece Çözülme. </li><li> Menadiyon küçük hacimleri (yaklaşık 50 mcL) içine aliquoted ve -70 saklanan 100% aseton 10 mM stok solüsyonu ° C olarak hazırlanmıştır. 10 ml çözülmüş XTT çözüm içeren bir tüp menodion, stok solüsyonu 1 mcL ekleyerek, kullanımdan hemen önce XTT / menodion çözüm hazırlayın. </li></ol></li><li> 37, 2 saat süreyle karanlıkta alüminyum folyo ve inkübe plakaları Kapak ° C </li><li> Plakalar ortaya çıkarın. Bir çok kanallı pipet kullanarak yeni bir mikrotiter plate ile ilgili kuyulara her iyi ve transfer renkli likit 80 mcL çıkarın. </li><li> Mikrotiter plate okuyucusu 490 nm&#39;de bir plaka (lar) ı okuyun. </li><li> Kolorimetrik okumaları bir kontrol antifungal ilaç yokluğunda oluşan biyofilm karşılaştırma (bu durumda 11 sütun için değerler% 50 veya% 80 azalma tespit edildiği antifungal konsantrasyonları sesil minimum inhibitör konsantrasyonları SMIC50 ve SMIC80 hesaplayın , negatif kontrol), sadece XTT içeren sütun 12 kuyulardan değerleri çıkarmak için de hatırlıyorum. SMIC sonuçları bir tablo (yani birden fazla antifungaller karşı birden fazla izolatları) veya alternatif olarak, her antifungal karşı izole her bir mantar için antifungal konsantrasyonu yüzde inhibisyonu karşı komplo grafik olarak sunulabilir olarak sunulabilir. </li></ol> Örnek: negatif kontrol değerleri çıkarılarak sonra, ortalama OD kontrolü sütun 11 oluşan biyofilm 1.32. SMIC50 az 1.32 x 50/100 = 0,66 Bu durumda, kolorimetrik okumaları&gt;% 50 azalma önde gelen düşük antifungal konsantrasyon. Aynı şekilde SMIC80, bu durumda daha az 1.32 x 20/100 = 0.264, kolorimetrik okumaları&gt;% 80 azalma önde gelen düşük antifungal konsantrasyon. 4. Temsilcisi Sonuçlar Şekil 1 C microphotograph albicans biyofilm ters bir mikroskop kullanılarak alınan bir 96 mikrotiter plate iyi bir alt kurdu. Şekil 2, her biri için bir C 11 biyofilm XTT kolorimetrik okumaları (OD 490 değerleri ) albicans vahşi tip suşu aynı 96 mikrotiter plate 8 farklı satırların her birinde kurdu. Şekil 3 amfoterisin B&#39;nin C&#39;ye karşı farklı konsantrasyonlarda faaliyet göstermektedir albicans biyofilm; oklar SMIC50 ve SMIC80 değerleri göstermektedir. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Şekil 1" /> Şekil 1. (A) Panel A, C inverted mikroskop bağlı bir kamera ile alınan bir microphotograph gösterir albicans biyofilm RPMI orta ve müteakip yıkamalar PBS ile aspirasyon sonra kuyunun altındaki kurdu. Tipik bir C (B) mikrografı albicans biyofilm taramalı elektron mikroskobu kullanılarak görüntülendi. Barlar panelleri sırasıyla A ve B için 100 mikron ve 10 mikron. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="Şekil 2" /> Şekil 2. Birden fazla eşdeğer C Oluşumu 96-iyi microtiter plakalar albicans biyofilm bir C oluşturduğu biyofilm XTT-azaltma testleri Kolorimetrik okumaları (OD 490 değerleri) microtiter plakalar kuyularda albicans yabani türü. Değerler aynı 96 mikrotiter plate 8 farklı satırların her birinde oluşan 11 bağımsız biyofilm içindir. Farklı satırlar için tek yönlü varyans analizi ve sapmaların homojenlik ve Bonferroni çoklu karşılaştırma testi sonrası Bartlett testi kullanılarak karşılaştırıldı. (P&gt; 0.05) her satır tüm çiftleri karşılaştırırken istatistiksel olarak anlamlı farklılıklar tespit edildi. <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Şekil 3" /> Şekil 3. C&#39;ye karşı antifungal duyarlılık testi tipik sonuçlar C biyofilm karşı farklı amfoterisin B konsantrasyonlarının etkinliği tipik sonuçlar betimleyen albicans biyofilm Grafik albicans vahşi tip süzün. Değerler ortalama yüzde kuyuları kontrol ile karşılaştırıldığında XTT azaltma deneyleri için kolorimetrik okuma olarak ifade edilir. SMIC50 ve SMIC80 değerleri oklarla gösterilir.

<ol>
	<li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activity+of+caspofungin+against+Candida+albicans+biofilms.">In vitro activity of caspofungin against Candida albicans biofilms.</a> Antimicrob Agents Chemother. 46, 3591-3596 (2002).</li><li>Hawser, S. P., Norris, H., Jessup, C. J., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t+etrazolium+hydroxide+(XTT)+colorimetric+method+with+the+standardized+National+Committee+for+Clinical+Laboratory+Standards+method+of+testing+clinical+yeast+isolates+for+susceptibility+to+antifungal+agents.">Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t etrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.</a> J Clin Microbiol. 36, 1450-1452 (1998).</li><li>Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+and+limitations+of+the+XTT+assay+in+studies+of+Candida+growth+and+metabolism.">Uses and limitations of the XTT assay in studies of Candida growth and metabolism.</a> J Clin Microbiol. 41, 506-508 (2003).</li><li>Kuhn, D. M., George, T., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+susceptibility+of+Candida+biofilms:+Unique+efficacy+of+amphotericin+B+lipid+formulations+and+echinocandins.">Antifungal susceptibility of Candida biofilms: Unique efficacy of amphotericin B lipid formulations and echinocandins.</a> Antimicrob. Agents Chemother. 46, 1773-1780 (2002).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+albicans+transcription+factor+Bcr1p:+a+regulator+of+cell-surface+genes+and+biofilm+formation.">C. albicans transcription factor Bcr1p: a regulator of cell-surface genes and biofilm formation.</a> Current Biol. 15, 1150-1155 (2005).</li><li>Ramage, G., Saville, S. P., Thomas, D. P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+biofilms:+an+update.">Candida biofilms: an update.</a> Eukaryot Cell. 4, 633-638 (2005).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., p, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45, 2475-2479 (2001).</li><li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+combinations+against+Candida+albicans+biofilms+in+vitro.">Antifungal combinations against Candida albicans biofilms in vitro.</a> Antimicrob Agents Chemother. 47, 3657-3659 (2003).</li><li>Ramage, G., VandeWalle, K., Bachmann, S. P., Wickes, B. L., Lopez-Ribot, J. 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JLL-R, antifungal ajanlar gelişmekte olan MicrobeHTS Technologies, Inc öz sermaye sahibi. MicrobeHTS Technologies, Inc bu çalışmalar için herhangi bir mali destek sağladı.

Burada XTT kullanarak biyofilm içinde hücrelerin metabolik faaliyetleri ölçen bir kolorimetrik yöntemi ile birleştiğinde Candida biyofilm oluşumu için, basit, hızlı, ekonomik ve yüksek tekrarlanabilir 96 mikrotiter plate modeli açıklanmaktadır . Bu biyofilm oluşumu için 96 iyi mikrotiter plate modeli C için geliştirilmiş. albicans, ancak diğer Candida türleri için kullanılabilir. ve diğer mantar organizmalar için kolayca adapte. Bu yöntem, birden fazla param...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Sabouraud-dextrose agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>To prepare plates for fresh subcultures of fungal isolates</td>
</tr>
<tr>
<td>YPD: Yeast peptone dextrose</td>
<td>&nbsp;</td>
<td>US Biological </td>
<td>Y2076</td>
<td>Medium for propagation of overnight liquid cultures</td>
</tr>
<tr>
<td>RPMI-1640 without sodium bicarbonate supplemented with L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-020-PB</td>
<td>Liquid medium for biofilm formation</td>
</tr>
<tr>
<td>Morpholinepropanesulfonic acid (MOPS)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP308</td>
<td>To buffer RPMI 1640</td>
</tr>
<tr>
<td>Phosphate buffered saline, PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>Buffer for washes</td>
</tr>
<tr>
<td>XTT sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X4251</td>
<td>See above for preparation instructions</td>
</tr>
<tr>
<td>Ringer's lactate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC0409-7953-09</td>
<td>For preparation of XTT solution</td>
</tr>
<tr>
<td>Menadione</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5625</td>
<td>Caution: hazardous by skin contact, inhalation or ingestion</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3595</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette and tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtiter plate reader</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

Candida albicans, şimdi ABD hastaneleri, üçüncü en sık görülen enfeksiyon bozukluğu olan hastalarda kandidiyazis ve genişleyen bir nüfus mantar enfeksiyonlarının en sık nedeni olmaya devam etmektedir . Kandidiyazis farklı tezahürleri biyofilm oluşumu, hem ev sahibi doku ve / veya tıbbi cihazlar (yani kateter) ile ilişkilidir. Biyofilm içinde hücrelerinin konağın immün yanıtları ve antifungallerin eylem korunur Biyofilm oluşumu, negatif klinik etkileri taşır. Biz C. oluşumu için, basit, hızlı ve güçlü bir in vitro model geliştirdik albicans biyofilm antifungal duyarlılık testi için de kullanılabilir 96 mikrotiter plakalar, biyofilm. Bu testte okuma, metabolik olarak aktif fungal biyofilm hücreleri tarafından XTT azaltılması (tetrazolium tuz) dayalı, kolorimetrik. Tipik bir deneyde, antifungal duyarlılık testi için ek bir 24 saat, biyofilm oluşumu için yaklaşık 24 saat sürer. Çünkü sadeliği ve yaygın olarak kullanılan laboratuar malzemeleri ve ekipman kullanımı, bu tekniğin, biyofilm araştırma democratizes ve antifungal duyarlılık testi mantar biyofilm standardizasyon doğru önemli bir adımı temsil etmektedir.

Watch this Scientific Journal Video about A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms at JoVE.com

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Biz oluşumu için basit, hızlı ve sağlam bir bir yöntem tarif<em&gt; Candida albicans</em&gt; Biyofilm içinde hücre antifungal duyarlılık testi 96 microtiter plakalar ve kendi programını kullanarak biyofilm.

İzleme Oluşumu ve Antifungal Duyarlılık Testi için 96 Şey microtiter Levha tabanlı Yöntemi Candida albicans Biyofilmler

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

44.5K Görüntüleme.  University of Texas San Antonio - UTSA. Biz oluşumu için basit, hızlı ve sağlam bir bir yöntem tarif Candida albicans Biyofilm içinde hücre antifungal duyarlılık testi 96 microtiter plakalar ve kendi programını kullanarak biyofilm.

Video: A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter Bulaşık Biyofilm Oluşumu Testi

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

İmmünoloji ve Enfeksiyon

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8-iyi Odası Slayt in vitro Biyofilm Oluşumu

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter Plate Format Broth Seyreltme Birinci ve İkinci Line İlaçları Mycobacterium tuberculosis kompleksi Antimikrobiyal Duyarlılık Testi

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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, ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Görselleştirme bir otomatik mikrosıvısal aygıtı kullanarak Candida albicans biyofilm oluşumu

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Derin Dermal enjeksiyon olarak bir Model Candida albicans deri enfeksiyonu histolojik analizler için

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Kırmızı ışık düzenlemek Candida albicans biyofilm büyüme ile günlük fototerapi

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Aydınlatma ve Görüntüleme Candida albicans Biyofilmler

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Candida albicans'ı İnhibe Etmek için Gül Bengal Aracılı Fotodinamik Terapi

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...