This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.

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The authors have nothing to disclose.

소형 형식으로 조류 성장. 현재 실험에서 복제가 증가 될 수 있도록 마이크로 타이 터 플레이트에서 1ml의 배양 부피 조류 배양의 소형화를 허용한다. 이 녹조류는 실험 내내 건강 보장하는 것이 중요하다; 조류의 영양 요건이 충족 될 수 있도록, 다양한 조류 매체를 평가하기 위해 마이크로 타이 터 플레이트 포맷을 사용하여, 성장 곡선 (도 2)를 수행한다. 또한,이?...

식물 플랑크톤의 생리는 전통적으로 원뿔 플라스크에 20 ㎖에서 carboys 1-7에서 5 L에 이르는 메조 규모의 실험에서 연구되어왔다. 각 시점에 대한 복제 샘플을 희생하는 것이 관리하기 어려운 실험 장치를 만들어으로이 실험 규모는 실험 모니터링을위한 서브 샘플링을 필요로한다. 줄이거 나 대량의 서브 샘플링 및 의사 - 복제의 제한을 제거한다 조류 생리학 실험 실험 용적을 소형화하여 일주 동일한 인큐베이터 공간을 사용하면서 기능은 독립적 인 실험의 수를 증가시키는. 마이크로 타이 터 플레이트 포맷은 가변 실험적 조건에서 조류를 조작 1ml의 배양 부피를 사용하여 조류 생물 검정을 위해 개발되었다. 실험이 작은 부피 복제물의 수가 증가 될 수 있도록 허용 복제에 의한 샘플 간의 감소 가변성 실험의 재현성을 증가(그림 2) 12 1백40일에 대한 실험 컨트롤 (즉, 무균 조류 배양)를 유지하면서 실험하고, 진정한 복제를 할 수 있습니다. 이 마이크로 타이 터 플레이트 형식은 쉽게 같은 실험 다양한 질문에 대한 적응 : 박테리아가 조류 호스트와 공생 중성 또는 병원성 상호 작용을해야합니까? 조류의 복합 자극의 추가 또는 독성인가? 이들 및 다른 문제는이 새로운 포맷을 사용하여 9-11 빠른 높은 처리량 방법으로 해결할 수있다. 48 웰 마이크로 타이 배양 플레이트는 각 1 ㎖를 잘 단일 시간 시점에서 샘플링 독립적 인 실험 장치가 될 수 있습니다. 다양한 매개 변수를 포함하여 1 ml의 볼륨에서 샘플링, 이에 국한되지 수 있습니다 WATER-펄스 진폭 변조 (WATER-PAM) 형광 측정 (재료 및 장비 표 참조) 1을 사용 엽록소 형광 및 광 화학적 매개 변수3. WATER-PAM의 형광 측정 조류 13의 실험을 모니터링하는 데 사용할 수있는 신속하고 비 침습적 방법이다. 14, 15 (WATER-PAM 4 ml의 볼륨 - - 2 중간에 희석 문화의 300 μL 150) 그것은 작은 문화 볼륨에서 광합성 효율과 광계 건강을 측정 할 수 있습니다. WATER-PAM의 형광 측정에 더하여,이 설정을 포함한 다른 다양한 파라미터를 측정하는데 사용될 수 있으나, 이에 제한되지 : 조류 세포 및 조류 세포 형태의 변화에​​ 부착 된 박테리아를 시각화하는 현미경; 단위 (CFU) 수를 형성하는 박테리아 식민지; 및 조류 세포 수 및 식별 주민들을위한 유동 세포 계측법.

<table><tbody><tr><td>10 cu. ft. Diurnal incubator (6012-1)</td><td>Caron Corporate</td><td>112310-6012-1-11</td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Nunc EasYFlask 25 cm&lt;sup&gt;2&lt;/sup&gt;, Vent/Close Cap, 7 ml working volume, 200/cs</td><td>Thermo Fisher Scientific</td><td>N156340</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>Multiwell TC Plates &amp;ndash; 48-well</td><td>BD Biosciences Discovery Labware</td><td>353078</td><td>&lt;a href=&quot;http://www.bdbiosciences.com/&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>P1000 Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F123602</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>P10mL Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F161201</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>Wide Orifice Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-468</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Ultrafine Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-470</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Finntip 10 ml [Vol: 1 - 10 ml]</td><td>Thermo Fisher Scientific</td><td>9402151</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>WATER-Pulse Amplitude Modulation (Water-ED)</td><td>Heinz Walz GmbH, Effeltrich, Germany</td><td>EDEE0232</td><td>&lt;a href=&quot;http://www.walz.com&quot;&gt;www.walz.com&lt;/a&gt;</td></tr><tr><td>15 mm diameter quartz glass cuvette (WATER-K)</td><td>Caron Corporate</td><td></td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Sodium chloride (crystalline/certified ACS), Fisher Chemical</td><td>Thermo Fisher Scientific</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>BD Difco Marine Broth 2216</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>BD Bacto Agar</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>L1 Medium Kit, 50 L</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>&lt;a href=&quot;http://www.ncma.bigelow.org&quot;&gt;www.ncma.bigelow.org&lt;/a&gt;</td></tr></tbody></table>

a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry

배양 실험 1-7에 걸쳐 서브 샘플링 될 수 있도록 미세 조류의 실험 조작을위한 종래의 방법은 대량 배양 (5 L 내지 20 ml)에 사용했다. 대량의 서브 샘플링 다양한 이유로 문제가 될 수있다 : 1)는 총 부피와 표면적의 변화 원인은 다음 실험 기간 동안 배양 체적비; 2) 의사 복제 (즉, 동일한 치료 플라스크 8에서 샘플)는 종종 오히려 진정한 복제물 (복제 치료에서 즉, 샘플링)보다 채용을 복제; 3) 실험 기간은 총 부피에 의해 제한된다; 4) 배양 무균 또는 일반적인 세​​균 미생물 오염은 일반적으로 서브 샘플링 동안 발생 장기 실험 동안 유지하기 어렵다. 미세 적정 플레이트의 사용은 최대 48 내의 별도의 처리 1 ㎖의 각각의 배양에 사용되는 복제 볼륨을 가능12.65 X 8.5 X 2.2 cm 판함으로써 실험 볼륨을 감소하고 치료를 서브 샘플링하지 않고 광범위한 복제를 허용. 9-11 스크리닝 세균성 조류 공동 배양, 조류 생리 테스트 및 독소 : 또한,이 기술은 실험을 포함한 다양한 포맷에 맞게 수정 될 수있다. 조류, 박테리아 및 / 또는 공동 문화와 개인 우물로 제한 등 다양한 실험 절차에 대한 샘플링,하지만 할 수 있습니다 WATER-펄스 진폭 변조 (WATER-PAM) 형광 측정, 현미경, 세균 집락 형성 단위 (CFU) 수와는 유동 세포 계측법. 마이크로 타이 터 플레이트 포맷 WATER-PAM 형광 측정의 조합은 광 화학적 수율 및 샘플, 높은 재현성과 낮은 가변성과 다른 광 화학적 파라미터의 다수의 신속한 측정이 가능하며 실험의 과정 속에 든 대형 유리 병 또는 원추형 플라스크를 서브 샘플링 많은 함정을 피할 .

WATER-PAM의 형광 측정 수치. 물 - 펄스 진폭 변조 (PAM) 형광 측정은 형광 (엽록소 콘텐츠에 대한 프록시) 및 조류 문화의 광합성 수율 (PSII 건강)를 결정하는 빠르고 효율적인 방법입니다. PAM WinControl 소프트웨어는 원시 데이터 값의 스프레드 시트 (다음 어두운 적응 조류 시료 기본적인 파라미터이다)를 생성한다 : 어두운 적응 세포의 F 0 = 형광 <p cl...

Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

작은 볼륨 생물 검정은 물 - 펄스 진폭 변조 (WATER-PAM) 형광 측정을 사용하여 세균 / 식물 플랑크톤의 공동 문화를 평가하는

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

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Universidad San Sebastian

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A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

10.5K 조회수.  University of Alberta. The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

비디오: A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

연구

면역학 및 감염병학

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

물 화학 및 해양 미생물에 필드 가이드 - 바다에서 보이지 않는 선수를 해명

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

환경과학

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 ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

microfluidic 비말 플랫폼 형광 검출 방법

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

생체공학

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Cytometry와 수생 Biofilms의 특성

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

수생 생태계에서 유기 탄소 흐름의 새로운 경로를 밝히기 위해 추적자로 형광으로 분류 된 박테리아

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

바이오 필름 및 서식지의 이질성의 공동 개발을 특성화하기위한 방법

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

천연 화합물 라이브러리의 탐험에 적합 안티 바이오 필름 분석 실험의 플랫폼

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, phototrophs also require illumination. Routinely, researchers seek to provide custom light supplies, i.e., vary the light intensity and time over which it is delivered. Such flexibility is difficult with standard benchtop lights. Usually, cultivation studies also require growth comparisons between experimental treatments. Frequently, growth is assessed over an extended duration, e.g., multiple times a day over a week-long trial. Manual measurements can be time-consuming and lack data resolution. Therefore, photobioreactors (PBRs) with automatic growth monitoring and customizable light supply are useful for replicated experiments with multiple treatments. The current work presents the design, construction, and operation of laboratory PBRs. The materials are easily sourced and relatively inexpensive. The design could be duplicated with moderate skill. Each structure has a footprint of ~40 cm2 and hosts three 1 L glass bottles for triplicate replication. Bottles rest upon platforms containing magnetic stirrers and are arranged vertically within a 1 m high and 15 cm diameter polyvinyl chloride (PVC) pipe. The pipe interior is lined with light-emitting diodes (LEDs). These LEDs produce continuous light intensities from 0&#8211;2400 &#181;mol photons m-2 s-1 of photosynthetically active radiation (PAR). Users design a custom lighting program. The light intensity can be adjusted each second or held constant for longer durations. Oxygen produced from photosynthesis exits each bottle via a one-way volumetric gas sensor. Software is used to record gas sensor data. The amount of oxygen produced can be correlated to biomass growth. If biomass samples are required, a syringe can be used to extract culture. The method is suited for microalgae grown with bicarbonate as the carbon source. These PBRs are valuable to a laboratory that requires replicated experiments, light regime flexibility, and continuous high-resolution growth data.

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

온라인 성장 측정 및 사용자 정의 가능한 조명 체계를 갖춘 실험실 광생물 반응기 운영

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, ...

생물학

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.