Name: paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis
Uploaded: 2018-03-23T14:00:00
Description: Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Эта работа была поддержана NSF карьеры премии 1553023 и NIH/NIAID премии R01AI103369.

1. рост Pseudomonas aeruginosa биоплёнки колонии
<ol><li>Подготовка среднего бислой пластин<ol>
<li>Подготовить Триптон 10 г/Л, 10 г/Л агар решение (см. Таблицу материалов) в деионизированной воде.</li>
		<li>Автоклав для 20 минут остыть до 50-60 ° C на водяной бане.</li>
		<li>Налейте 45 мл раствора агар Триптон к...

<ol>
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Образцы тканей парафин встраивание и тонко секционирования представляет собой классический гистологический метод, который позволяет изображения микро морфологических структур и широко используется на эукариотических ткани и с некоторым успехом был применен для микробиологических...

Большинство микробы способны формы биопленки, общины клеток скрепленных собственного производства матриц. Биопленки можно выращивать во многих типах физических установок, с различных режимов предоставления питательных веществ и субстрата. Конкретных анализов для биопленки, как правило, дают воспроизводимости многоклеточные структуры, и общей архитектуры наблюдаются филогенетически разнообразных видов на макроскопическом уровне или сообщества. Когда микробы выращиваются как колоний на твердом носителе под атмосферу, макроскопической морфология передает сведения о емкости для производства матрицы и часто коррелирует с2,другие черты в 1,3. Внутренняя архитектура микробной колоний может также предоставить подсказки относительно конкретных биопленки химия и физиология, но было трудно охарактеризовать. Последние приложений загрузки криостата и cryosectioning методов бактериальных колоний позволили изображений и визуализации определенных функций на беспрецедентной резолюции 4,5,6. Однако исследования с тканей животных показали, что парафин встраивание обеспечивает улучшенный сохранение морфологии, когда по сравнению с загрузки криостата 7 и был использован для визуализации бактерий в тканях 8,9. Поэтому мы разработали протокол для фиксации, внедрение парафин и тонкие секционирование колонии микробной биопленки. Здесь мы будем описывать подготовки Pseudomonas aeruginosa PA14 колонии биопленки шлифов 10,11, но мы также успешно применен этот метод к биопленки, образованный бактерии Pseudomonas synxantha, Сенная палочка, и холерный вибрион12.
Процесс биоплёнки парафин встраивание и тонко секционирования следует простая логика. Во-первых биоплёнки помещены в слое агар для сохранения морфология во время обработки. Во-вторых заключенная биоплёнки погружали в фиксатор чтобы crosslink макромолекул и сохранить микроморфологии. Эти затем обезвоживается с алкоголем, очищаются с более неполярных растворителей и затем проникли с жидкого парафина. После того, как проникли, образцы встроены блоки воск для разрезания. Секции являются вырезать, монтируется на слайдах и затем Регидратированные для того, чтобы вернуть их в более родного государства. С этого момента они могут окрашенных или покрыты в среде установки для микроскопического анализа.
Этот протокол производит шлифов микробной биоплёнки подходит для гистологического анализа. Колонии биопленки подструктурами видны, когда шлифов, подготовлен с использованием этого метода отражаются на световой микроскопии. Биопленки можно также выращивать на СМИ содержащие флуоресцентные пятна специфических для индивидуальных особенностей или витражи на шаге регидратации, непосредственно до монтажа (шаги 9.5-9,6). Наконец микробы могут быть спроектированы для получения флуоресцентных белков в учредительных или регулируется моды, позволяя в situ отчетности распределения или гена выражения ячейки в этих общинах. Мы использовали эти методы для определения глубины биопленки колонии, распределение ячеек, матрица распределения, шаблонов роста и экспрессии генов пространственно-временных.

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paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis

Секционирование через встраивание парафин методика широко установленным в eukaryotic систем. Здесь мы предоставляем метод для фиксации, внедрение и секционирование нетронутыми микробной колонии биоплёнки с помощью перфузии парафина. Адаптировать этот метод для использования на колонии биопленки, мы разработали методы для поддержания каждого образца на подложке ее роста и ламинирование с агар слоя и Добавлено Лизин в фиксирующие решение. Эти оптимизации улучшить образца удержание и сохранение функции микроморфологических. Подготовленный таким образом образцы поддаются тонких секционирование и изображений на свет, флуоресценции и передачи электронной микроскопии. Мы применили эту технику в колонии биоплёнки Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilisи Vibrio cholerae. Высокий уровень детализации, видимой в образцах, созданный этим методом, в сочетании с репортером штамм инженерных или использование определенных красителей, может предоставить интересные идеи в физиологии и развитие микробных сообществ.

Этот метод создает биопленки, тонко секции которой морфологических особенностей и зон экспрессии генов может быть imaged DIC, флуоресцентной микроскопии и ТЕА. В то время как DIC изображений с помощью 40 X цель погружения нефти может быть достаточно, чтобы показать некоторые ...

Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Встраивание парафин и тонкий секционирование колонии микробной биоплёнки для микроскопического анализа

Мы описываем фиксации, внедрение парафин и тонкого резания методы для колонии микробной биопленки. В подготовленных образцах биопленки каркаса и репортер выражение шаблоны могут быть визуализированы при микроскопии.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

The overall goal of this procedure is to generate paraffin embedded biofilm sections that can be used to assess the distribution of gene expression as reported by fluorescent protein production within intact native morphologies. This method can help answer key questions in the study of microbial communities such how the expression of specific genes in the production of exopolymeric substances distributed in the context of native biofilm architecture. The main advantage of this technique is that is allows for analysis of gene expression within the preserved morphology of biofilm cross-sections while also being amenable to downstream treatments such as staining of distinct features and high-resolution imaging such confocal or electron microscopy.After preparing agar tryptone solution according to the text protocol, use a 50-milliliter conical tube to pour 45 milliliters of the solution into a 100-millimeter by 100 millimeter square dish. Allow the agar to solidify for approximately 20 to 30 minutes. Pour a second 15-millimeter layer on top of the first layer, then let it solidify overnight, and if necessary, use a lint-free tissue to remove condensation from the lids.Next, to spot colony biofilms, after growing an isolated single P.Aeruginosa colony from a frozen stock according to the text protocol, pipette 2.5 to 10 microliters of cell suspension onto a medium bi-layer plate. Colonies may be incubated in the dark at 25 degrees Celsius in 80 to 100%relative humidity for up to four days. Gently pour 15 milliliters of the agar solution over the growth medium and colony and allow the agar to form a gel at room temperature for five minutes.Then, use a sharp razor blade to cut a square, three-layered chuck with the colony laminated between the top two layers. If preparing multiple samples, ensure that each chuck is cut to a comparable size. Gently remove any excess agar from the colony containing chuck.Then, wet the flat head of a spatula in PBS or water and gently insert it between the top and bottom layers of agar. Immediately transfer the laminated colony into an embedding cassette labeled with a chemical-resistant marking pen. Place the embedding cassette into a glass slide mailer containing previously prepared fixative.Process the samples. Use 1X PBS to wash them twice for one hour each. For best results, automate reagent homogenization using the low setting on the spin function of an automatic tissue processor.To embed the samples, fill a wax mold with molten paraffin wax heated to 55 degrees Celsius, then use a heated flat spatula to quickly transfer the infiltrated chuck from the embedding cassette into the wax mold ensuring that the chuck rests parallel to the base of the mold. Allow the wax to solidify overnight at four degrees Celsius. Samples molded in solid wax may be stored indefinitely at this temperature.Once the wax has solidified, excise the sample from the mold and use a razor blade to trim excess wax from around the sample. Leave some wax extending from one end of the sample that can be used to clamp it into the microtome. It is important to trim the wax-embedded sample such that its shape facilitates the collection of smooth linear ribbons.Small imperfections in trimming can produce artifacts in the ribbon and compromise the integrity of the section. Next, heat a water bath to 42 degrees Celsius. Then, clamp the sample into the microtome with the surface of the colony oriented perpendicularly to the edge of the blade.Trim the sample in 50-micrometer intervals until the desired plane of the colony has been reached from which sections will be collected using a microtome set to a sectioning velocity of 75 to 80 RPM and a clearance angle of six to 10 degrees. Cut a ribbon of the desired number of 10-micrometer-thick sections. Then, use a fine-tipped paint brush to detach the ribbon from the blade.Now, with forceps or a drop of water at the tip of a Pasteur pipette, gently transfer the ribbon to the water bath. Immediately insert a slide into the water bath and position it below the ribbon at a 45-degree angle. Touch the narrow edge of the ribbon to the slide just below the frosted label.The ribbon should adhere. Pull the slide out of the water bath and adjust the angle so it's perpendicular to the surface of the water allowing the ribbon to lay flat against the slide along its length. Avoid trapping excess water beneath the ribbon.Gently stand the slide onto an absorbent lint-free tissue wicking excess water from the section. Then, lay the slide on a paper towel and allow it to dry in the dark at room temperature overnight. Heat a leveled hot plate to 45 degrees Celsius and place the slide on the plate for 30 to 60 minutes.The wax will become semi-molten and flatten against the slide. Gently lift the slide from the hot plate and lay it flat onto a smooth level room temperature surface for approximately one minute until the wax solidifies. Ensure that the molten wax does not pull to either side of the slide.With the samples inside a glass slide mailer, de-wax the slides in four washes of clearing agent for five minutes each. Use a Buchner aspirator to remove solution between washes. Use 100%ethanol to wash the slides three times for one minute each.Then, after rehydrating the slides according to the text protocol, immediately mount the sections in a tris-buffered mounting medium and apply a coverslip. The overlay agar around the section does not react with the fixative, so it does not adhere to the chard slide. This agar can dislodge during rehydration and damage the section, so care should be taken not to agitate the solution during rehydration.Allow the mounting medium to polymerize at room temperature overnight. Once polymerized, use clear nail polish to seal the coverslip to the slide. Finally, store the sealed slides indefinitely in the dark at four degrees Celsius.While DIC imaging using a 40 times oil immersion objective can be sufficient to show some morphological features of biofilm thin sections, fluorescence microscopy of strains engineered to constitutively express fluorescent proteins provides enhanced visualization of cellular distribution within the samples. As shown in these panels, images of individual sections can stitched together to generate a cross-section of the entire colony and provides context for the localization of structural features within the overall morphology at the macroscopic level. Using a strain engineered to express fluorescent protein under the control of a specific promoter enables the visualization of the distribution of gene expression.In addition, colonies can be grown on medium containing dyes, or dyes can be added post-sectioning to stains'specific polysaccharides. Finally, samples can also be prepared and imaged at higher resolution using transmission electron microscopy as presented here. It's important when attempting this procedure to be mindful of the samples'vagility and of the potential to introduce fluorescent and structural artifacts either through fixation, sectioning or rehydration which may influence your interpretation of the data.This procedure allows for a quantitative image analysis such as correlating the expression profiles of fluorescent reporters with morphological features and the Z-axis of a biofilm. This protocol can be applied to diverse species of biofilm-forming microbes with medical and industrial significance such as Pseudomonas aeruginosa, Vibrio cholerae and Bacillus subtilis. After watching this video, you should have a good understanding of how to grow and prepare colony biofilms for sectioning via paraffin embedding.

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Колония Формирование Cell (CFC) Пробирной по правам гемопоэтических клеток

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Одноклеточный анализ Сенная Bacillus Биопленки с помощью флуоресцентной микроскопии и проточной цитометрии

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Рост Микобактерий туберкулеза Биопленки

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Выделение и анализ генома Единого Вирионы использованием &quot;Единое геномики Вирус&quot;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

Логометрический визуализации внеклеточного рН в зубных биопленок

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Новый метод для качественного многомасштабном анализа Бактериальный биопленок на нитчатых конидий с помощью конфокальной и электронной микроскопии

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Извлечение Hemocytes из личинки Drosophila melanogaster микробной инфекции и анализа

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Дополнительное использование микроскопических методов и флуоресценции Чтение в изучении Cryptococcus-Amoeba Взаимодействия

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Применение Live Cell Imaging и крио-электронной томографии для устранения пространственно-временной особенностей системы Legionella pneumophila Dot/Icm Secretion

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Анализ липидного состава микобактерий методом тонкослойной хроматографии

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...