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 of first layer. Let solidify overnight, removing condensation from lids if necessary by wiping with a lint-free tissue.</li>
	</ol>
	</li>
	<li>Spotting Colony Biofilms
	<ol>
		<li>Streak the strains of interest from freezer stocks onto LB agar plates13 and incubate in the dark for 12-16 h at 37 &#176;C and 80-100% relative humidity.</li>
		<li>For each strain or replicate, use a single colony to inoculate 2 mL of LB and incubate in the dark for 12-16 h at 37 &#176;C with shaking at 250 rpm.</li>
		<li>Sub-culture by diluting 1:100 into fresh LB and incubating in the dark for 2.5-3 h at 37 &#176;C with shaking at 250 rpm.</li>
		<li>Measure optical density using a spectrophotometer and adjust with sterile LB to achieve a cell suspension with an OD500 nm of ~0.5.</li>
		<li>Depending on the desired colony size, pipet 2.5-10 &#181;L of cell suspension onto a medium-bilayer plate (prepared in step 1.1) and allow the spot to dry for ~20 min. Leaving the petri lid ajar near an open flame can facilitate drying.</li>
		<li>Incubate spot colonies at 25 &#176;C and 80-100% relative humidity, in the dark for up to 4 days.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Fixative Solution
<ol>
	<li>Prepare the fixative solution on the day of sample harvesting. Dilute 37% formaldehyde (FA) in 1x PBS to a final concentration of 4% FA. 
	CAUTION: FA is volatile and toxic. Wear protective equipment and work in a well-ventilated fume-hood.</li>
	<li>Immediately before use, dissolve L-lysine hydrochloride in 4% FA (prepared in step 2.1) at room temperature to a final concentration of 50 mM L-lysine HCl.</li>
</ol>
3. Direct Application of Fixative to the Colony Biofilm [Optional]
NOTE: We have found that biofilm morphology is best preserved when the agar overlay is added before fixation. However, this step also constitutes a change in environmental conditions that could affect gene expression. Fluorescent reporter expression patterns should therefore be verified using a separate protocol in which the fixation step is carried out before the addition of the agar overlay, as described here.
<ol>
	<li>On the day of fixation, and working in a well-ventilated fume-hood, pipette the fixative prepared in step 2 directly to the agar surface around the colony&#39;s edge, allowing it to reach the colony&#39;s periphery without submerging it. Apply only as much fixative as is needed to fully surround the colony, approximately 500 &#181;L. Allow the fixative to diffuse into the colony and the surrounding agar.</li>
	<li>Once the fixative has been fully absorbed into the colony and surrounding media, about 20-30 min, and residual FA is no longer visible on the plate, repeat step 3.1. Allow this fixative to diffuse into the colony and the surrounding agar.</li>
	<li>Once the fixative has been fully absorbed into the colony and surrounding media repeat step 3.1, this time applying fixative to the surface of the colony directly. Allow this fixative to diffuse into the colony and the surrounding agar, proceeding to step 4 only after all fixative has been absorbed and is no longer visible on the plate.</li>
</ol>
4. Overlaying Colonies with Agar
<ol>
	<li>On the day of fixation, prepare a 10 g/L agar solution in deionized water and autoclave for 10-20 minutes to dissolve. Cool in a water bath to 50 &#730;C.</li>
	<li>Gently pour 15 mL of the agar solution over the growth medium and colony. Allow the agar to form a gel at room temperature for 5 min (Figure 1B).</li>
	<li>Use a sharp razor blade to cut a square, 3-layer chuck with the colony laminated between the top two layers. If preparing multiple samples, ensure that each chuck is cut to a comparable size.</li>
	<li>Gently remove excess agar from the colony-containing chuck.</li>
	<li>To lift the two upper layers of agar (containing the colony) away from the bottom layer of the chuck (Figure 1C), wet the flat head of a spatula in 1x PBS or water and gently insert between the top and bottom layers. The laminated colony should separate from the bottom layer. Be careful not to distort or bend the laminated colony when lifting it from its base.</li>
</ol>
5. Fixation
<ol>
	<li>Working in a well-ventilated fume-hood, immediately transfer the laminated colony (i.e., 2-layer chuck) into an embedding cassette (see Table of Materials), labeled with a chemical-resistant marking pen. Place the embedding cassette into a glass slide mailer (see table of materials) containing the fixative prepared in steps 2.1-2.2. Make sure there are no air bubbles trapped inside the embedding cassette.</li>
	<li>Incubate the sample in fixative overnight in the dark at room temperature.</li>
</ol>
6. Sample Processing: Buffer Wash, Dehydration, Clearing, and Infiltration
<ol>
	<li>After overnight fixation, wash the sample twice in 1x PBS for 1 h each. For best results, automate reagent homogenization using the spin function of an automatic tissue processor (see table of materials) set to a low setting. If no processor is available, processing can be run manually.</li>
	<li>Follow the buffer wash by dehydration through a graded series of ethanol (EtOH) dilutions in 1x PBS (25%, 50%, 70%, 95%) for 1 h each at room temperature.</li>
	<li>Dehydrate in three washes of 100% EtOH for 1 h each at room temperature.</li>
	<li>Clear the dehydrated sample in three washes of 100% orange oil-based clearing agent (see table of materials) for 1 h each at room temperature.</li>
	<li>Infiltrate the cleared sample twice with molten paraffin wax (see Table of Materials), heated to 55&#730;C, for 2 h each.</li>
</ol>
7. Sample Embedding
<ol>
	<li>Fill a wax mold with molten paraffin wax heated to 55 &#730;C. 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. Avoid applying excessive pressure onto the chuck. Wax molds may be custom 3-D printed, or are commercially available (see Table of Materials).</li>
	<li>Allow wax to solidify overnight at 4 &#730;C. Samples molded in solid wax may be stored indefinitely at 4 &#730;C.</li>
	<li>Once the wax has solidified, excise the sample from the mold and trim excess wax from around the sample with a razor blade. Leave excess wax extending from one end of the sample, parallel to the plane of sectioning, that can be used to clamp it into the microtome (see Table of Materials). Also leave a sheath of wax, approximately 1-mm thick, around the sample and smooth its faces.</li>
</ol>
8. Sectioning
<ol>
	<li>Heat a water bath to 42 &#730;C. Clamp the sample into the microtome with the surface of the colony oriented perpendicularly to the edge of the blade (see Table of Materials).</li>
	<li>Trim the sample in 50-&#181;m intervals until the desired plane of the colony has been reached, from which sections will be collected.</li>
	<li>Cut a ribbon of the desired number of 10-&#181;m thick sections using a microtome set to a sectioning velocity of 75-80 rpm and a clearance angle of 6-10&#730;. Use a fine-tipped paintbrush to detach the ribbon from the blade.</li>
	<li>Using a drop of water at the tip of a Pasteur pipette (preferred) or forceps, gently transfer the ribbon to the water bath. Be careful to avoid trapping air bubbles under the section.</li>
	<li>Immediately insert a slide into the water bath and position it below the ribbon at a 45&#730; angle.</li>
	<li>Touch the narrow edge of the ribbon to the slide, just below the frosted label. The ribbon should adhere.</li>
	<li>Pull the slide out of the water bath, adjusting the angle to become perpendicular to the surface of the water, and allowing the ribbon to lay flat against the slide along its length. Avoid trapping excess water beneath the ribbon.</li>
	<li>Gently stand the slide onto an absorbent lint-free tissue, wicking excess water from the section.</li>
	<li>Lay the slide on a paper towel, and allow it to dry at room temperature overnight in the dark. Slides can be stored indefinitely at 4 &#730;C in the dark.</li>
</ol>
9. Heat-fixing, Rehydration, and Mounting
<ol>
	<li>Heat a leveled hot plate to 45 &#730;C. Heat the slide for 30-60 min. The wax will become semi-molten and flatten against the slide.</li>
	<li>Gently lift the slide from the hot plate and lay it flat onto a smooth, leveled, room temperature surface, using care to ensure that the molten wax does not pull to either side of the slide, until the wax solidifies (~1 min).</li>
	<li>Using a glass slide mailer, de-wax slides in four washes of clearing agent for 5 min each, using an B&#252;chner aspirator to remove solution between washes.</li>
	<li>Wash the slides three times in 100% EtOH for 1 min each.</li>
	<li>Rehydrate the slides in a graded series of ethanol in the reverse order of processing (95%, 70%, 50%, and 25%) for 1 min each followed by two 1-min washes in 1x PBS.</li>
	<li>Immediately mount the sections in a Tris-buffered mounting medium (see Table of Materials) and apply a coverslip, avoiding the introduction of air bubbles. Allow mounting medium to polymerize overnight at room temperature.</li>
	<li>Once polymerized, seal the coverslip to the slide using clear nail polish. Sealed slides may be stored indefinitely in the dark at 4 &#730;C.</li>
</ol>

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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">5 3/4&#34; Pasteur pipette</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">13-678-6A</td>
			<td style="width:577px;">Purchased from univeristy biostores&#160;</td>
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			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">A7777</td>
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			<td style="width:191px;">Pyrex</td>
			<td style="width:161px;">5340</td>
			<td>Purchased from univeristy biostores&#160;</td>
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			<td style="width:191px;">VWR</td>
			<td style="width:161px;">103051-182</td>
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			<td style="width:191px;">********</td>
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			<td height="42" style="height:42px;width:276px;">TRIS-buffered Mounting Medium (w/ DAPI)&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">50 247 04</td>
			<td>Manufacturer: Electron Microscopy Sciences</td>
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			<td height="21" style="height:21px;width:276px;">Embedding Mold&#160;</td>
			<td style="width:191px;">********</td>
			<td style="width:161px;">********</td>
			<td>3D printed in-house</td>
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			<td height="42" style="height:42px;width:276px;">Embedding Mold (commercial)&#160;</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:161px;">70182</td>
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			<td height="21" style="height:21px;width:276px;">Ethanol 200P</td>
			<td style="width:191px;">Decon Labs, Inc.&#160;</td>
			<td style="width:161px;">2701</td>
			<td>Purchased from univeristy biostores&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Fine-tipped Brush</td>
			<td style="width:191px;">********</td>
			<td style="width:161px;">********</td>
			<td>Store bought, paint brush</td>
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			<td height="21" style="height:21px;width:276px;">Glass Coverslips 60x22mm</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">12-519-21C</td>
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			<td height="21" style="height:21px;width:276px;">Glass Rehydration Mailer&#160;</td>
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			<td style="width:161px;">21043</td>
			<td>20 slide mailer&#160;</td>
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			<td style="width:191px;">Fisher Scientific</td>
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			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">BP386 100</td>
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			<td>p10 (Fisher Scientific, 02 707 469), p200 (VWR, 89079-474), p1250 (VWR, 89079-486)</td>
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			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
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			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Razor Blades&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Slide Warmer&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Sodium chloride&#160;</td>
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			<td style="width:161px;">0241-1KG</td>
			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Sodium phosphate&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">BDH9296.500</td>
			<td>,Component of phosphate buffered saline, prepared in-house&#160;</td>
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			<td height="21" style="height:21px;width:276px;">Suprafrost Histology Slides&#160;</td>
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			<td style="width:161px;">12-544-2</td>
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			<td height="21" style="height:21px;width:276px;">Tissue Flotation Water Bath&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">NC0815797</td>
			<td>Manufacturer: Ted Pella, Vendor Catalog # 28156-B</td>
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			<td height="21" style="height:21px;width:276px;">Automatic Tissue Processor&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">813160U/Q#00009061</td>
			<td>Model: STP120 Tissue Processor</td>
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			<td height="21" style="height:21px;width:276px;">Tryptone&#160;</td>
			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">T9012</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Yeast extract</td>
			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">Y9010</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

paraffin-embedding-thin-sectioning-microbial-colony-biofilms-for

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Immunology and Infection

14.2K Views.  Columbia University. 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.

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

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 leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

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

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 constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

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

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. 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

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

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 are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

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

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

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

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by 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

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

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

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

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

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

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

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

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

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

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

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

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

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...