Biofilme relacionadas ao trabalho no laboratório é financiado por verbas numeradas e R21DE017294 R21AI080930 do Instituto Nacional de Pesquisa Dental e Craniofacial e do Instituto Nacional de Alergia e Doenças Infecciosas (para JLL-R.). O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do NIDCR, o NIAID ou o NIH.

1. Preparação de C. albicans C. albicans é um microorganismo 1/BSL1 Risk Group. Lembre-se sempre ao bom uso de técnicas assépticas / estéril para trabalhar com este microorganismo e seguir os procedimentos institucionais para o descarte adequado de materiais de risco biológico. <ol><li> Prepare uma cultura overnight de C. albicans em YPD médio (Yeast peptonada Dextrose) líquido através da inoculação de uma única colônia de C. albicans em 25 mL de YPD. </li><li> Incubar a cultura em um shaker orbital (cerca de 180 rpm) a 30 ° C durante a noite. Mais C. albicans cepas vai crescer como células de levedura sob essas condições. </li><li> Centrifugar as culturas durante a noite (cerca de 3.000 rpm, 5-10 minutos), lave as células duas vezes com PBS estéril, e ressuspender o sedimento final em cerca de 20-25 mL de meio RPMI 1640 tamponado com 165 mM de ácido morpholinepropanesulfonic a 7,0 pH e pré-aquecido a 37 ° C (a partir de agora este meio é referido simplesmente como &quot;RPMI 1640&quot;). </li><li> Contagem de células utilizando um hemocitômetro. Após a contagem, prepare uma suspensão de células em uma densidade final de 1,0 x 10 6 células / mL em RPMI 1640. Nota: Como as células têm uma tendência a se agregar, é importante vortex vigorosamente entre as lavagens e antes de pipetar. </li></ol> 2. Configurando o de 96 poços da placa de microtitulação para a formação do biofilme <ol><li> Usando uma pipeta multicanal adicionar 100 mL de C. albicans suspensão em poços selecionados dos 96 poços da placa de microtitulação (-s). Não adicione células para poços na coluna 12, como estes servirão como controles negativos. </li><li> Cobrir a placa de microtitulação inteiro com sua tampa original, vedação com parafilme, coloque dentro de uma incubadora e incubar por 24 horas a 37 ° C. A duração da incubação pode ser adaptado para o projeto experimental específico. Por exemplo, é possível examinar a cinética de formação de biofilme ao longo de um período de 24-48 horas de semeadura placas múltiplas e processamento de cada placa em momentos diferentes (ie 2, 4, 8, 12, 24 e 48 h) </li><li> Após a formação de biofilme, usando uma pipeta multicanal aspirar o meio com cuidado de não tocar e interromper os biofilmes que se formaram em cada um dos poços. </li><li> Usando uma pipeta multicanal placas lavar três vezes em PBS estéril (200 mL por poço). Alternativamente, use uma lavadora automática de microtitulação. Entre as lavagens, e particularmente após a última lavagem, escorrer as placas em posição invertida por blotting com toalhas de papel para remover qualquer PBS residual. Neste ponto biofilmes formados no fundo dos poços deve ser claramente visível mesmo a olho nu e também pode ser visualizado através de um microscópio invertido (Figura 1). Biofilmes está agora pronto para ser processada para os testes de testes de susceptibilidade antifúngica. (Se o principal objetivo do experimento é avaliar a extensão da formação de biofilme, as placas estão prontas para serem processadas utilizando o método colorimétrico. Para isso, o reagente XTT / menadiona (consulte a Etapa 3 abaixo) pode ser adicionado ea cor resultante ler usando um leitor de placas de microtitulação). </li></ol> 3. Testes de Susceptibilidade antifúngica de Biofilmes <ol><li> A partir de soluções estoque ou em pó, prepare uma solução de trabalho em meio RPMI 1640 de cada antifúngico a ser testado. Típico altas concentrações são 1.024 mcg / mL para fluconazol, e 16 mcg / mL para ambos anfotericina B e caspofungina. Outras concentrações podem ser usados ​​para diferentes agentes. </li><li> Usando uma pipeta multicanal, adicione 200 mL da alta concentração de trabalho de antifúngicos aos poços correspondentes na coluna 1 de cada placa de microtitulação contendo biofilmes de fungos. </li><li> Adicionar 100 mL de RPMI 1640 em cada poço nas colunas 2 a 10. </li><li> Adicionar 100 mL de RPMI 1640 para poços na coluna 11. </li><li> Remover 100 mL de agente antifúngico dos poços da coluna 1 e adicione aos poços adjacentes na coluna 2 (já contendo 100 mL de meio). </li><li> Misture o conteúdo bem pipetando cima e para baixo para realizar uma diluição de duplicação de série, e retire as pontas. </li><li> Repita se movendo para a direita até os poços da coluna 10, após o qual o volume de 100 mL finais dos poços da coluna 10 após a mistura é descartado. Desta forma, uma série de diluições de seu agente (-s) de interesse foram criados, a partir de mais concentrada em poços da coluna 1 para o menos concentrado em poços da coluna 10. Biofilmes incontestado na coluna 11 servirá como controle positivo, e poços vazios na coluna 12 servirão como controles negativos. </li><li> Cobrir as placas com suas tampas, vedação com parafilme e incubar por 24 -48 h a 37 ° C. </li><li> Após o período de incubação, lavar os pratos como na Etapa 2,4 antes (3 x PBS). </li><li> Usando uma pipeta multicanal adicionar 100 mL de XTT / menadiona solução para cada poço contendo um biofilme pré-lavados, bem como para poços de controlo negativo para a medição de fundo XTT-colorimétrico níveis. <ol><li> O XTT é preparado como uma solução saturada, 0,5 g / L de lactato Ringer estéril, a PBS ou salina, que precisa ser esterilizada por filtração, usando um filtro de 0,22 mM tamanho de poros. A solução XTT é sensível à luz, por isso deve ser coberto com papel alumínio durante a preparação. Depois de preparado e esterilizada por filtração, alíquota, em 10 mL volumes de trabalho, e armazenar a -70 ° C. Proteger os tubos de luz usando uma folha de alumínio. Descongele somente como tubos que forem necessárias para uma experiência especial pouco antes de usar. </li><li> Menadiona é preparado como uma solução estoque 10 mM em acetona 100%, aliquotado em volumes menores (cerca de 50 mL) e armazenados a -70 ° C. Prepare a solução XTT / menadiona pouco antes de usar, adicionando 1 mL da solução estoque de menadiona a um tubo contendo 10 mL da solução XTT descongelado. </li></ol></li><li> Cobrir as placas em papel alumínio e incubar no escuro por 2 h, a 37 ° C. </li><li> Descubra as placas. Usando uma pipeta multicanal remover 80 mL do sobrenadante resultante cor de cada bem e transferir para os poços correspondente de uma nova placa de microtitulação. </li><li> Leia a placa (-s) em um leitor de placas de microtitulação a 490 nm. </li><li> Calcular a concentração inibitória mínima sésseis SMIC50 e SMIC80, que são as concentrações antifúngicas em que uma diminuição de 50% ou 80% em leituras colorimétricas são detectados em comparação com o controle de biofilmes formados na ausência de drogas antifúngicas (neste caso, os valores para a coluna 11 , lembre-se também para subtrair valores de controles negativos de poços na coluna 12 XTT contendo apenas). SMIC resultados podem ser apresentados como uma tabela (ou seja, isola múltiplas contra antifúngicos múltipla) ou, alternativamente, os resultados individuais para cada fúngicas isolar uns contra os antifúngicos pode ser apresentado como um gráfico plotando inibição por cento versus concentração de antifúngico. </li></ol> Exemplo: Depois de subtrair os valores no controle negativo, o OD média de biofilmes controle formado na coluna 11 é 1.32. O SMIC50 é a menor concentração antifúngicos levando a uma redução&gt; 50% em leituras colorimétricas, neste caso, menos de 1,32 x 50/100 = 0,66. Da mesma forma, o SMIC80 é a menor concentração antifúngicos levando a uma redução&gt; 80% em leituras colorimétricas, neste caso, menos de 1,32 x 20/100 = 0,264. 4. Resultados representante A Figura 1 mostra uma microfotografia de um C. albicans biofilme formado no fundo de um poço em uma placa de microtitulação de 96 poços realizadas em microscópio invertido. A Figura 2 mostra XTT-colorimétrico leituras (OD 490 valores) para cada um dos 11 biofilmes de C. albicans cepa selvagem formado em cada uma das 8 linhas diferentes da mesma placa de microtitulação de 96 poços. A Figura 3 mostra a atividade de anfotericina B em diferentes concentrações contra C. albicans biofilmes; setas indicam SMIC50 e SMIC80 valores. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Figura 1" /> Figura 1. (A) Painel A mostra uma microfotografia tiradas com uma câmera acoplada a um microscópio invertido de um C. albicans biofilme formado no fundo do poço após a aspiração de meio RPMI e lavagens subsequentes com PBS. (B) A micrografia de um típico C. albicans biofilme visualizados através de microscopia eletrônica de varredura. Barras são 100 mm e 10 mm para os painéis A e B, respectivamente. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="Figura 2" /> Figura 2. Formação de múltiplas equivalente C. albicans biofilmes em placas de 96 poços de microtitulação. leituras colorimétrico (OD 490 valores) de XTT-redução de ensaios de biofilmes formados por um C. albicans tipo selvagem em poços de placas de microtitulação. Os valores são de 11 biofilmes independente formada em cada uma das 8 linhas diferentes da mesma placa de microtitulação de 96 poços. Resultados para as linhas diferentes foram comparados por meio da análise de variância e pelo teste de Bartlett para homogeneidade de variâncias e comparação múltipla de Bonferroni pós-teste. Diferenças estatisticamente significativas foram observadas quando se comparam todos os pares de linhas entre si (P&gt; 0,05). <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Figura 3" /> Figura 3. Os resultados típicos dos testes de susceptibilidade antifúngica contra C. albicans biofilmes Gráfico. retratando os resultados típicos da eficácia de diferentes concentrações de anfotericina B contra biofilmes de C. albicans cepa selvagem. Os valores estão expressos em porcentagem média leituras colorimétrico para XTT redução de ensaios em relação ao controle de poços. SMIC50 e SMIC80 valores são indicados por setas.

<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. 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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. 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JLL-R possui capital em MicrobeHTS Technologies, Inc., que está desenvolvendo antifúngicos. MicrobeHTS Technologies, Inc. não forneceu apoio financeiro para estes estudos.

Aqui nós descrevemos um método simples, rápido, econômico e altamente reprodutível 96 modelo da placa de microtitulação bem para a formação de biofilmes de Candida juntamente com um método colorimétrico que mede a atividade metabólica de células dentro do biofilme usando XTT. Este modelo de 96 placa de microtitulação bem para a formação de biofilme foi originalmente desenvolvido para C. albicans, mas pode ser usado para outras Candida spp. e facilmente adaptado para outros org...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
		</table>
		</div>

a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

Candida albicans continua a ser a causa mais freqüente de infecções fúngicas em uma população em expansão de pacientes comprometidos e candidíase é agora o terceiro mais comum de infecção nos hospitais dos EUA. Diferentes manifestações da candidíase são associados com a formação de biofilme, tanto em tecidos do hospedeiro e / ou dispositivos médicos (cateteres, por exemplo). Formação de biofilme carrega negativas implicações clínicas, como as células dentro do biofilme estão protegidos das respostas imune do hospedeiro e da ação de antifúngicos. Nós desenvolvemos um modelo simples, rápido e robusto in vitro para a formação de C. biofilmes albicans utilizando 96 poços de microtitulação-pratos, que também pode ser usado para testes de susceptibilidade antifúngica do biofilme. A leitura deste ensaio é colorimétrico, baseado na redução de XTT (um sal de tetrazólio) por células metabolicamente ativas biofilme fúngicas. Um experimento típico dura cerca de 24 h para a formação de biofilme, com 24 h adicionais para testes de susceptibilidade antifúngica. Devido à sua simplicidade e à utilização de materiais de laboratório comumente disponíveis e equipamento, esta técnica democratiza pesquisa biofilme e representa um passo importante para a padronização dos testes de susceptibilidade antifúngica de biofilmes de fungos.

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

Nós descrevemos um método simples, rápido e robusto para a formação de<em&gt; Candida albicans</em&gt; Biofilmes usando 96 placas de microtitulação bem e sua utilidade em testes de susceptibilidade antifúngica de células dentro de biofilmes.

A 96 Bem microtitulação Método da Placa com base para a Formação Monitoramento e Teste de susceptibilidade antifúngica de Candida albicans Biofilmes

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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Research

JoVE Journal

Immunology and Infection

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

44.5K visualizações.  University of Texas San Antonio - UTSA. Nós descrevemos um método simples, rápido e robusto para a formação de Candida albicans Biofilmes usando 96 placas de microtitulação bem e sua utilidade em testes de susceptibilidade antifúngica de células dentro de biofilmes.

Vídeo: 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.

Dish microtitulação Assay formação de biofilme

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

Imunologia e Infecção

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

In vitro a formação de biofilme em um slide Câmara de 8 poços

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.

Teste de susceptibilidade antimicrobiana do complexo Mycobacterium tuberculosis da Droga primeira e segunda linha de diluição em caldo em um formato de placa de microtitulação

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.

Preparação de uma Cultura Pellet sangue para rápida identificação bacteriana e Antibiótico Teste à Susceptibilidade

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.

Um método de microdiluição em caldo de referência para Dalbavancin<em&gt; In Vitro</em&gt; Teste à Susceptibilidade de bactérias que crescem aeròbia

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.

Visualização da formação de biofilmes de Candida albicans , usando um dispositivo Microfluidic automatizada

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.

Profunda dérmica injeção como um modelo de Candida albicans de infecção da pele para análise histológica

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.

Diária fototerapia com luz vermelha para regular crescimento de biofilme de Candida albicans

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.

Esclarecendo e Imagem Candida albicans Biofilmes

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.

Terapia fotodinâmica mediada por Rose Bengala para inibir a 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, ...