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.

צמיחת אצות בפורמט מיניאטורי. המזעור של תרבויות אצות למיליליטר 1 נפח תרבות בצלחת microtiter מאפשר לשכפול בניסוי להיות מוגבר. זה חשוב כדי להבטיח את האצה היא בריאה לאורך ניסוי; לבצע עקומת צמיחה (איור 2), תוך שימוש בפורמט צלחת microtiter ?...

הפיסיולוגיה הפיטופלנקטון באופן מסורתי למדה בניסויים טווינה-סולם הנעים בין 20 מיליליטר בצלוחיות חרוטי עד 5 L בcarboys 1-7. בקנה מידה ניסיוני זה דורש subsampling לניטור ניסוי, כהקרבת דגימות לשכפל לכל נקודת זמן יוצרת הגדרת ניסוי בלתי ניתנת לניהול. היכולת להגדיל את מספר ניסויים בלתי תלויים תוך השימוש באותו מרחב חממה יומית על ידי miniaturizing הנפח הניסיוני לניסויי פיזיולוגיה אצות יהיה להפחית או לבטל את המגבלות של subsampling ופסאודו-שכפול מכמויות גדולות. פורמט צלחת microtiter פותח עבור bioassays אצות באמצעות 1 מיליליטר נפח תרבות לניסוי מניפולציה אצות בתנאים משתנים. נפח קטן וניסיוני זה מאפשר למספר החזרות להיות מוגבר, מגדיל שחזור ניסוי בשל השתנות ירד בין דגימות ולשכפלניסויים, ומאפשר שכפול אמיתי תוך שמירה על בקרת ניסוי (כלומר, תרבויות אצות axenic) עבור 140 ימים (איור 2) 12. פורמט צלחת microtiter זה מותאם בקלות למגוון רחב של שאלות ניסיוניות, כגון: האם יש חיידק אינטראקציה סימביוטית, ניטראלית או פתוגניים עם מארח האצות שלה? האם התוספת של מגרה מתחם או רעילה לאצה? שאלות אלו ואחרות ניתן לטפל באופן תפוקה גבוהה מהיר באמצעות פורמט חדש זה 9-11. צלחת תרבות microtiter 48 היטב מאפשרת לכל 1 מיליליטר גם להיות התקנה ניסיונית עצמאית שתדגם בנקודת זמן אחת. ניתן לדגום פרמטרים שונים מזה 1 מיליליטר כולל נפח, אך לא רק: הקרינה כלורופיל ופרמטרי פוטו באמצעות fluorometry במים Pulse-Amplitude-אופנן (WATER-PAM) (ראה חומרים וציוד שולחן) 13. Fluorometry במים PAM הוא טכניקה מהירה ולא פולשנית, שניתן להשתמש כדי לפקח על ניסויים שבוצעו עם אצות 13. זה מאפשר מדידה של יעילות פוטוסינתזה ובריאות PSII מקטן נפח תרבות (150-300 μl של התרבות בדילול מלא במדיום ל2-4 מיליליטר נפח מים-PAM) 14,15. בנוסף לfluorometry במים PAM, התקנה זו יכולה לשמש כדי למדוד מגוון של פרמטרים אחרים, כולל, אך לא מוגבלת ל: מיקרוסקופיה לדמיין את החיידקים מצורפים לתאים ושינויים במורפולוגיה של תאי אצות אצות; מושבת חיידקים יוצרת יחידת ספירה (CFU); וcytometry זרימה לספירת תאי אצות ותת-אוכלוסיות זיהוי.

<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

שיטות מקובלות למניפולציה ניסויית של microalgae יש מועסקי כמויות גדולות של התרבות (20 מיליליטר עד 5 L), כך שהתרבות יכולה להיות subsampled לאורך הניסוי 1-7. Subsampling של כמויות גדולות יכול להיות בעייתי מכמה סיבות: 1) זה גורם לשינוי בשטח פן הנפח והכולל: יחס נפח של התרבות במהלך הניסוי; 2) פסאודו-שכפול (כלומר, לשכפל דגימות מאותו בקבוק הטיפול 8) לעתים קרובות מועסק ולא משכפל אמיתי (דגימה כלומר, מטיפולים לשכפל); 3) משך הניסוי מוגבל על ידי הנפח הכולל; ו 4) תרבויות axenic או חיידקי חיידקים הרגילים קשים לשמור במהלך ניסויים לטווח ארוך כזיהום מתרחש בדרך כלל במהלך subsampling. השימוש בצלחות microtiter מאפשרת כרכי 1 מיליליטר תרבות שישמשו לכל לשכפל, עם עד 48 טיפולים נפרדים בתוךצלחת סנטימטר 12.65 x 8.5 x 2.2, ובכך להקטין את הנפח הניסיוני ומאפשר לשכפול נרחב ללא subsampling כל טיפול. בנוסף, טכניקה זו יכולה להיות שונה כדי להתאים מגוון רחב של פורמטים ניסיוניים כוללים: שיתוף תרבויות חיידקים-אצות, בדיקות פיזיולוגיה של אצות, ורעלן הקרנת 9-11. בארות בודדות עם אצה, חיידק ו / או שיתוף תרבויות ניתן לדגום לנוהלי מעבדה רבים, כולל, אך לא מוגבלת ל: WATER-Pulse-Amplitude-אופנן (WATER-PAM) fluorometry, מיקרוסקופיה, יחידת מושבת חיידקי יוצרים (CFU) רוזנים וcytometry זרימה. השילוב של fluorometry פורמט צלחת microtiter ומים-PAM מאפשר מדידות מהירות מרובות של תשואת פוטו ופרמטרים אחרים פוטו עם שונות נמוכות בין דגימות, שחזור גבוה וימנע את החסרונות רבים של subsampling בַּקבּוּק או בקבוק חרוטי במהלך ניסוי .

קריאות fluorometry במים PAM. במים Pulse-Amplitude-אופנן (PAM) fluorometry היא שיטה מהירה ויעילה כדי לקבוע את הקרינה (proxy עבור תוכן כלורופיל) ותשואת פוטוסינתזה (בריאות PSII) של תרבויות אצות. תוכנת PAM WinControl יוצרת גיליון אלקטרוני של ערכי נתונים גולמיים ל( להלן ?...

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.

מבדק נפח קטן להערכה בקטריאלי / הפיטופלנקטון Co-תרבות שימוש במים-Pulse-Amplitude-אופנן (WATER-PAM) Fluorometry

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

a-small-volume-bioassay-to-assess-bacterialphytoplankton-co-culture

Universidad San Sebastian

Research

JoVE Journal

Environment

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

10.5K Views.  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.

Video: 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.

Co-מודלים של תרבות Pseudomonas aeruginosa Biofilms גדל על תאים חיים Airway האדם

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

Immunology and Infection

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.

להתיר את השחקנים הנעלמת באוקיינוס ​​- מדריך שדה לכימיה מים והימי מיקרוביולוגיה

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

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.

Biofilm microtiter תבשיל גיבוש 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 ...

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.

שיטות גילוי הקרינה לפלטפורמות אגל microfluidic

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

Bioengineering

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.

אפיון Biofilms ימיים עם Cytometry זרימה

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.

בקטריה מתויג חיידקים כנותב כדי לחשוף מסלולים הרומן של זרימת פחמן אורגנית במסגרת אקולוגית מימיים

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.

שיטות לאפיון Co-הפיתוח של Biofilm ובית גידול הטרוגניות

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.

פלטפורמה של מבחנים אנטי biofilm המתאים לחקירת ספריות תרכובת טבעיות

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.

הפעלת פוטו-ביוריאקטורים מעבדתיים עם מדידות צמיחה מקוונות ומשטרי אור הניתנים להתאמה אישית

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

Biology

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.

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 photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

מדידת פוטופיזיולוגיה של השלב המצורף של קולאקיום sp. על ידי פלואורומטר קצב חזרה מהירה מסוג Cuvette

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

מבדק נפח קטן להערכה בקטריאלי / הפיטופלנקטון Co-תרבות שימוש במים-Pulse-Amplitude-אופנן (WATER-PAM) Fluorometry

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Co-מודלים של תרבות Pseudomonas aeruginosa Biofilms גדל על תאים חיים Airway האדם

להתיר את השחקנים הנעלמת באוקיינוס - מדריך שדה לכימיה מים והימי מיקרוביולוגיה

Biofilm microtiter תבשיל גיבוש Assay

שיטות גילוי הקרינה לפלטפורמות אגל microfluidic

אפיון Biofilms ימיים עם Cytometry זרימה

בקטריה מתויג חיידקים כנותב כדי לחשוף מסלולים הרומן של זרימת פחמן אורגנית במסגרת אקולוגית מימיים

שיטות לאפיון Co-הפיתוח של Biofilm ובית גידול הטרוגניות

פלטפורמה של מבחנים אנטי biofilm המתאים לחקירת ספריות תרכובת טבעיות

הפעלת פוטו-ביוריאקטורים מעבדתיים עם מדידות צמיחה מקוונות ומשטרי אור הניתנים להתאמה אישית

מדידת פוטופיזיולוגיה של השלב המצורף של קולאקיום sp. על ידי פלואורומטר קצב חזרה מהירה מסוג Cuvette

מבדק נפח קטן להערכה בקטריאלי / הפיטופלנקטון Co-תרבות שימוש במים-Pulse-Amplitude-אופנן (WATER-PAM) Fluorometry

Summary

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Chapters in this video

Co-מודלים של תרבות Pseudomonas aeruginosa Biofilms גדל על תאים חיים Airway האדם

להתיר את השחקנים הנעלמת באוקיינוס ​​- מדריך שדה לכימיה מים והימי מיקרוביולוגיה

Biofilm microtiter תבשיל גיבוש Assay

שיטות גילוי הקרינה לפלטפורמות אגל microfluidic

אפיון Biofilms ימיים עם Cytometry זרימה

בקטריה מתויג חיידקים כנותב כדי לחשוף מסלולים הרומן של זרימת פחמן אורגנית במסגרת אקולוגית מימיים

שיטות לאפיון Co-הפיתוח של Biofilm ובית גידול הטרוגניות

פלטפורמה של מבחנים אנטי biofilm המתאים לחקירת ספריות תרכובת טבעיות

הפעלת פוטו-ביוריאקטורים מעבדתיים עם מדידות צמיחה מקוונות ומשטרי אור הניתנים להתאמה אישית

מדידת פוטופיזיולוגיה של השלב המצורף של קולאקיום sp. על ידי פלואורומטר קצב חזרה מהירה מסוג Cuvette

להתיר את השחקנים הנעלמת באוקיינוס - מדריך שדה לכימיה מים והימי מיקרוביולוגיה