실험실에서 Biofilm 관련 작업은 부여에 의해 자금 치과 &amp; Craniofacial 연구 및 알레르기 및 전염병 (JLL - R 있습니다.)에 대한 국립 연구소의 국립 연구소에서 R21DE017294 및 R21AI080930 번호가 있습니다. 내용은 전적으로 저자의 책임이며 반드시 NIDCR, NIAID 또는 NIH의 공식 견해를 대변하지 않습니다.

1. C.의 준비 albicans C. albicans는 위험 그룹 1/BSL1 미생물입니다. 항상이 미생물과 함께 일을 잘 무균 / 살균 기술을 사용하여 생물 학적 물질의 적절한 처리를위한 제도적 절차를 따르도록 기억 해요. <ol><li> C.의 야간 문화를 준비 C.의 단일 식민지 inoculating하여 YPD (효모 펩톤 덱스 트로 오스) 액체 매체 albicans YPD 25 ML에 albicans. </li><li> 30 ° C 하룻밤에 궤도 쉐이크 (180 RPM)에서 문화를 품다. 대부분의 C. albicans 변종이 조건 하에서 효모 세포로 성장합니다. </li><li> 야간 문화를 (약 3,000 RPM, 5-10분) 원심 분리기, 멸균 PBS로 두 번 세포를 씻어하고, 산도 7.0과 미리 예열을 165 MM morpholinepropanesulfonic의 산성과 버퍼 RPMI 1640 매체의 20-25에 대해 ML의 마지막 펠릿을 resuspend 37 ° C가 (이 매체에 지금부터 간단하게 &quot;RPMI 1640&quot;이라고합니다). </li><li> hemacytometer를 사용하여 셀을 계산합니다. 계산 후, 1.0 X 10 6 세포 / RPMI 1640에 ML의 최종 농도에서 세포 현탁액을 준비합니다. 참고 : 세포가 집계하는 경향을 가지고 있기 때문에,이 세탁 사이 pipetting 전에 적극적으로 와동하는 것이 중요합니다. </li></ol> 2. Biofilm의 형성을위한 96 - 웰 플레이트 Microtiter 설정 <ol><li> 멀티채널 피펫을 사용하면 C. 100 μL를 추가 96 - 웰 플레이트 microtiter (- S)의 선택 우물에 albicans 서스펜션. 이들은 부정적인 조절 역할을하므로, 열 12 우물에 전지를 추가하지 마십시오. </li><li> 원래 뚜껑이있는 전체 microtiter 접시, parafilm와 인감, 37 24 H에 대한 보육 및 품어 안으로 장소 ° C. 표지 부화의 길이는 특정 실험 설계에 적용할 수 있습니다. 다른 시간 지점 (즉 2, 4, 8, 12, 24 및 48 H)에서 여러 개의 접시를 시딩 각 플레이트를 처리하여 48 시간은 됐어 - 예를 들어, 24의 기간 동안 biofilm 형성의 속도론을 조사 할 수 있습니다 </li><li> biofilm 형성 후, 멀티채널 피펫을 사용하는 것은 신중로 우물 각 형성 biofilms를 만지거나 방해하지 매체를 대기음. </li><li> 멸균 PBS (물론 당 200 μL)에서 멀티채널 피펫 세척 접시 세 번 사용합니다. 또는 자동 microtiter 접시 세척기를 사용합니다. 세척 사이에, 특히 마지막 세척 후, 잔여 PBS를 제거하기 위해 종이 타월과 모래 바닥으로 바뀌 위치에 접시를 물기. 이 시점에서 우물의 바닥에 형성 biofilms도 육안으로 분명히 볼 수 있어야하며 거꾸로 현미경 (그림 1)를 사용하여 시각하실 수 있습니다. Biofilms 지금 antifungal 자화율 시험 assays을 위해 처리될 준비가 된 것입니다. (실험의 주요 목적은 biofilm 형성의 범위를 평가하는 경우에는 번호판 colorimetric 방법을 사용하여 처리할 준비가 된 것입니다.이 경우, XTT / 메나 디온 시약은 (아래 3 단계 참조) 결과 색상을 추가하고 수 ) microtiter 플레이트 판독기를 사용하여 읽어보십시오. </li></ol> 3. Biofilms의 Antifungal 자화율 테스트 <ol><li> 재고 솔루션 또는 분말에서, 테스트되는 각 antifungal의 RPMI 1640 배지에서 작업 솔루션을 준비합니다. 전형적인 높은 농도는 1,024 fluconazole에 대한 μg / ML, 16 amphotericin B와 caspofungin 모두 μg / ML 있습니다. 다른 농도가 다른 에이전트에 사용할 수 있습니다. </li><li> 멀티채널 피펫을 사용하여 곰팡이 biofilms를 포함하는 각 microtiter 접시 컬럼의 1에 해당 우물에 antifungal의 높은 작업 농도 200 μL를 추가합니다. </li><li> 10 열 2 각 잘하는 RPMI 1640 100 μL를 추가합니다. </li><li> 열 11 우물에 RPMI 1,640 100 μL를 추가합니다. </li><li> 열 1의 우물에서 antifungal 요원 100 μL를 제거 및 열 2의 인접 우물 (이미 매체의 100 μL를 포함)에 추가할 수 있습니다. </li><li> 잘 직렬 두 배로 희석을 수행하고 위아래로 pipetting하여 내용을 혼합하고, 도움말을 제거합니다. </li><li> 오른쪽 혼합 후 열 10 우물에서 최종 100 μL 볼륨이 삭제되는 열의 후 10 우물, 이동 때까지 반복합니다. 이러한 방법으로 관심의 에이전트 (- S)의 dilutions을 배로 일련의이 생성되었습니다; 대부분 열 10 우물에 최소 농축 열 1의 우물에 집중되어에서. 열 11 도전받지 않은 biofilms 긍정적인 컨트롤로 제공되며, 열에 12 빈 우물은 부정적인 제어 역할을합니다. </li><li> 37 그들의 뚜껑, 24 -48 H에 대한 parafilm과 부화로 도장과 번호판을 커버 ° C. </li><li> 잠복기 후 (3 X PBS) 이전 단계 2.4로 접시를 씻으십시오. </li><li> 멀티채널 피펫을 사용하면 배경 X의 측정에 대한 부정적인 제어 우물에 잘 미리 씻어 biofilm을 포함하는 각각의뿐만 아니라 XTT / 메나 디온 솔루션 100 μL를 추가TT - colorimetric 수준. <ol><li> XTT는 0.5 0.22 μm의 - 기공 크기의 필터를 사용하여 필터 소독해야 멸균 링거스 락테이트, PBS 또는 호수에 G / L에서 포화 솔루션으로 준비가되어 있습니다. XTT 솔루션은 민감한 빛 때문에, 그것은 준비 중에 알루미늄 호일으로 덮여 있어야합니다. -70 번에 준비 및 필터 - 소독, 10 ML 작업 볼륨으로 나누어지는하고, 저장 ° C. 알루미늄 호일을 사용하여 빛을에서 튜브를 보호합니다. 사용하기 직전 특정 실험에 필요한 많은 튜브로만 해동. </li><li> 메나 디온은 작은 볼륨 (약 50 μL)로 aliquoted과 -70에 저장된 100 % 아세톤에 10 MM 주식 솔루션 ° C.으로 준비가되어 있습니다 해동 XTT 솔루션 10 ML을 포함하는 튜브에 메나 디온의 주식 솔루션 1 μL를 추가하여, 사용하기 직전 XTT / 메나 디온 솔루션을 준비합니다. </li></ol></li><li> 37 2 H 동안 짙은 어둠 속에서 알루미늄 호일과 부화에 번호판을 커버 ° C. </li><li> 번호판을 발견. 멀티채널 피펫을 사용하면 새로운 microtiter 접시의 해당 우물에 각각 잘하고 전송에서 발생하는 색깔 뜨는의 80 μL를 제거합니다. </li><li> 490 NM에서 microtiter 플레이트 판독기에서 접시를 (- S) 참조하십시오. </li><li> colorimetric 판독의 50 % 80퍼센트 감소가 antifungal 약물의 부재에 형성된 제어 biofilms에 비해 (이 경우 항목에 11 값을 감지되는 antifungal 농도의 고착의 최소 억제 농도 SMIC50과 SMIC80를 계산 ,) 만 XTT를 포함하는 열에 12 우물에서 부정적인 컨트롤에서 값을 빼야하는 것도 기억나요. SMIC 결과는 표 (여러 antifungals에 대해 즉, 여러 개의 격리) 혹은 다른 방법으로, 각 antifungal에 대한 격리 각 곰팡이에 대한 검색 결과가 antifungal 농도 비교 %의 억제를 계획하여 그래프로 제시 수 있듯이 제시하실 수 있습니다. </li></ol> 예 : 대조군의 값을 공제한 후, 열 11 형성된 제어 biofilms의 평균 OD가 1.32입니다. SMIC50이 경우보다 1.32 X 100분의 50가 = 0.66에서 colorimetric 낭독의&gt; 50 % 감소로 이어지는 가장 낮은 농도 antifungal입니다. 마찬가지로, SMIC80이 경우보다 1.32 X 1백분의 20가 = 0.264에서 colorimetric 낭독의&gt; 80 % 감소로 이어지는 가장 낮은 농도 antifungal입니다. 4. 대표 결과 그림 1은 C.의 microphotograph을 보여줍니다 albicans biofilm은 거꾸로 현미경을 사용하여 촬영 96 잘 microtiter 접시에있는 우물의 바닥에 형성. 그림 2는 C. 11 biofilms 각 XTT - colorimetric 신호 (OD 490 값을)를 보여줍니다 albicans 야생 유형 스트레인 같은 96 잘 microtiter 접시의 8 가지 행을 각 형성. 그림 3은 C.에 대해 서로 다른 농도에서 amphotericin B의 활동을 보여줍니다 albicans biofilms; 화살표 SMIC50과 SMIC80 값을 나타냅니다. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="그림 1" /> 그림 1. (A) 패널은 C.의 거꾸로 현미경에 부착된 카메라를 사용하여 촬영한 microphotograph을 보여줍니다 albicans biofilm은 RPMI 매체와 PBS와 후속 세탁의 열망 후 우물의 바닥에 형성. 전형적인 C의 (B) 현미경 albicans biofilm은 스캐닝 전자 현미경을 사용하여 시각. 바 패널 각각 A와 B 100 μm의 10 μm의 수 있습니다. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="그림 2" /> 그림 2. 여러 동등한 C.의 형성 96 - 웰 microtiter 접시에 albicans biofilms. C. 형성 biofilms의 XTT - 감소 assays에서 Colorimetric 판독 (OD 490 값이) microtiter 접시의 우물에서 albicans 야생 입력합니다. 값은 동일한 96 잘 microtiter 접시 8 다른 행의 각 형성 11 독립 biofilms위한 것입니다. 다른 행에 대한 결과는 편도 분산 분석 및 변동의 균질성 및 Bonferroni의 다중 비교 후 시험에 바틀 테스트를 사용하여 비교했다. (P&gt; 0.05) 서로의 모든 행을 쌍을 비교하면 아무 통계적으로 의미있는 차이는 언급되지 않았다. <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="그림 3" /> 그림 3. C. 반대 antifungal 자화율 시험의 일반적인 결과 C.의 biofilms에 대한 다른 amphotericin B로 농도의 효능의 전형 결과를 묘사한 albicans biofilms. 그래프 albicans 야생 유형 변형. 값은 우물을 제어에 비해 XTT - 감속 assays 평균 퍼센트 colorimetric 수치로 표현됩니다. SMIC50과 SMIC80 값은 화살표로 표시됩니다.

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JLL - R은 antifungal 요원을 개발 MicrobeHTS 테크놀로지 주식 회사에 지분을 소유하고 있습니다. MicrobeHTS 테크놀로지 주식 회사는 이러한 연구에 대한 재정 지원을 제공하지 않습니다.

여기 XTT를 사용하여 biofilm 내에서 세포의 대사 활동을 측정 colorimetric 방법으로 결합 캔디다 biofilms의 형성을위한 단순하고 신속하고 경제적이며 높은 재현성 96 웰 플레이트 microtiter 모델을 설명합니다. biofilm 형성을위한이 96 잘 microtiter 플레이트 모델은 원래 C. 위해 개발되었습니다 albicans하지만 다른 캔디다 종에 사용될 수 있습니다. 쉽게 다른 곰팡이 유기체에 대한 ?...

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<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

캔디다 albicans 지금은 미국 병원에서 세 번째로 가장 일반적인 감염 위험 환자와 칸디다 증의 확대 인구의 곰팡이 감염의 가장 빈번한 원인 남아있다. 칸디다 증의 발현은 다른 호스트 조직 및 / 또는 의료 기기 (즉 카테터)에 두 biofilm 형성과 관련이 있습니다. biofilms 내에서 세포가 면역 반응 호스트로부터 antifungals의 행동으로부터 보호되면서 Biofilm 형성은 부정적인 임상 영향을 수행합니다. 우리는 C. 형성을위한 간단하고 신속하고 강력한에서 체외 모델을 개발 또한 biofilm antifungal 자화율 시험에 사용할 수있는 96 잘 microtiter - 접시를 사용 albicans의 biofilms. 이 분석의 판독은 metabolically 활성 곰팡이 biofilm 세포에 의해 XTT의 감소 (tetrazolium 소금)을 기반으로, colorimetric입니다. 전형적인 실험은 antifungal 자화율 시험에 대한 추가 24 H와 biofilm 형성에 대한 약 24 H 걸립니다. 때문에 단순하고 일반적으로 사용 가능한 실험실 재료 및 장비의 사용이 기술은 biofilm 조사를 democratizes 및 곰팡이 biofilms의 antifungal 자화율 테스트 표준화 향한 중요한 단계를 나타냅니다.

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

우리는 형성을위한 간단하고 신속하고 강력한 방법을 설명<em&gt; 캔디다 albicans</embiofilms 내에서 세포의 antifungal 자화율 시험에 96 잘 microtiter 접시와 유틸리티를 사용하여&gt; biofilms.

모니터링 형성과 Antifungal 자화율 테스트를위한 96 자 Microtiter 플레이트 기반 방법 캔디다 albicans Biofilms

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

a-96-well-microtiter-plate-based-method-for-monitoring-formation

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 조회수.  University of Texas San Antonio - UTSA. 우리는 형성을위한 간단하고 신속하고 강력한 방법을 설명 캔디다 albicans biofilms.

비디오: 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 디시 Biofilm 형성 분석

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

연구

면역학 및 감염병학

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 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

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 플레이트 형식의 맛을 희석하여 첫 번째와 두 번째 라인 의약품 Mycobacterium 결핵의 복합 항균 자화율 테스트

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.

빠른 세균의 식별 및 항생제 감수성 테스트를위한 혈액 문화 펠렛의 제조

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.

Dalbavancin에 대한 참조 국물 Microdilution 방법<em&gt; 체외</em호기성 박테리아의 성장&gt; 감수성 테스트

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.

Candida albicans 자동 미세 장치를 사용 하 여에서 Biofilm 형성의 시각화

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.

깊은 피부 주입으로는 모델 조직학 분석 Candida albicans 피부 감염의

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.

규제 Candida albicans Biofilm 성장에 붉은 빛으로 매일 광선 요법

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.

칸디다 알비칸스 바이오필름 의 명확화 및 이미징

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.

로즈 벵골 매개 광역학 치료 칸 디다 알비칸을 억제하기 위해

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