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

This work was supported by NSF CAREER AWARD 1553023 and NIH/NIAID award R01AI103369.

1. Growth of Pseudomonas aeruginosa Colony Biofilms
<ol>
	<li>Preparation of Medium-Bilayer Plates
	<ol>
		<li>Prepare a 10 g/L tryptone, 10 g/L agar (see Table of Materials) solution in deionized water.</li>
		<li>Autoclave for 20 min. Cool to 50-60 &#176;C in a water bath.</li>
		<li>Pour 45 mL of the agar-tryptone solution into a 100 mm x 100 mm square dish (see Table of Materials) using a 50-mL conical tube. Allow the agar to solidify (~20-30 min). Pour a second, 15-mL layer on top...

<ol>
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Paraffin-embedding and thin-sectioning tissue samples is a classic histological technique that enables imaging of micro-morphological structures and is commonly used on eukaryotic tissues, and has been applied with some success to microbial samples8,9. While cryoembedding allows for strong retention of endogenous and immunofluorescent signal, paraffin embedding is generally preferable as it provides better preservation of morphology16. In ...

Most microbes have the capacity to form biofilms, communities of cells held together by self-produced matrices. Biofilms can be grown in many types of physical setups, with various regimes of nutrient and substrate provision. Specific assays for biofilm formation tend to yield reproducible multicellular structures, and common architectures are observed for phylogenetically diverse species at the community or macroscopic level. When microbes are grown as colonies on solid medium under an atmosphere, macroscopic morphology conveys information about the capacity for matrix production and often correlates with other traits 1,2,3. The internal architecture of microbial colonies can also provide clues regarding biofilm-specific chemistry and physiology, but has been difficult to characterize. Recent applications of cryoembedding and cryosectioning techniques to bacterial colonies have enabled imaging and visualization of specific features at unprecedented resolution 4,5,6. However, studies with animal tissue have shown that paraffin embedding provides superior preservation of morphology when compared to cryoembedding 7 and has been used to visualize bacteria in tissues 8,9. We have therefore developed a protocol for fixation, paraffin embedding, and thin sectioning of microbial colony biofilms. Here, we will describe the preparation of Pseudomonas aeruginosa PA14 colony-biofilm thin sections 10,11, but we have also successfully applied this technique to biofilms formed by the bacteria Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae12.
The process of paraffin-embedding and thin-sectioning biofilms follows a simple logic. First, the biofilms are encased in a layer of agar to preserve morphology during processing. Second, the encased-biofilms are submerged in a fixative to crosslink macromolecules and preserve micromorphology. These are then dehydrated with alcohol, cleared with a more non-polar solvent, and then infiltrated with liquid paraffin wax. Once infiltrated, the samples are embedded into wax blocks for sectioning. Sections are cut, mounted on slides, and then rehydrated in order to return them to a more native state. From this point, they can be stained or covered in mounting medium for microscopic analysis.
This protocol produces thin sections of microbial biofilms suitable for histological analysis. Colony biofilm substructures are visible when thin sections prepared using this method are imaged by light microscopy. Biofilms can also be grown on media containing fluorescent stains specific for individual features or stained at the rehydration step, immediately prior to mounting (steps 9.5-9.6). Finally, microbes can be engineered to produce fluorescent proteins in a constitutive or regulated fashion allowing in situ reporting of cell distribution or gene expression within these communities. We have used these methods to determine colony biofilm depth, cell distribution, matrix distribution, growth patterns, and spatiotemporal gene expression.

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

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

This method generates biofilm thin-sections wherein distinct morphological features and zones of gene expression can be imaged by DIC, fluorescence microscopy, and TEM. While DIC imaging using a 40X oil immersion objective can be sufficient to show some morphological features (Figure 2E), we have found that fluorescence microscopy of strains engineered to constitutively express fluorescent protein provides enhanced visualization of cell distribution within th...

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

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

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

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Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

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

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

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'

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

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

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

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

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

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

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

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...