The authors would like to acknowledge Otterbein University for Undergraduate Student Research Fellowships and Student Research Fund Awards to GVK and SGK; and the Otterbein Professor Gilbert E. Mills Memorial Endowed Sabbatical Program Award and The Department of Biology and Earth Science Faculty Research and Scholarship Endowed Award to JAB. The Bert and Jane Horn Endowed Student Research Fund in the Sciences was awarded to GVK and SGK. The authors would also like to gratefully acknowledge Duquesne University funded Undergraduate Research Program Fellowships for GVK and SGK.&#160;Former Juniata College undergraduate research students, Ryan Johnson and Lindsey Draper contributed the microscopy images for Figures 2 and 3, respectively.

1. Prepare Instruments, Culture Media, Solutions, and Petri Dishes
<ol>
	<li>Autoclave toothpicks in a 50 mL beaker covered with aluminum foil to keep sterile.</li>
	<li>Sterilize all dry goods that will be used (tips, sticks, glassware, etc.) in an autoclave. 
	NOTE: Caution! Follow all safety directions for the autoclave model used. Autoclaves pose a risk for explosions, and burns from steam and contact with hot surfaces, materials, and media. For autoclave safety, view &#34;Proper Use of Autoclaves&#34;34.</li>
	<li>Prepare the MS agar (Mannitol Soya Flour (SFM))14 to grow cultures.
	<ol>
		<li>Add 5 g of soya flour and 5 g of agar to 1 L flask.</li>
		<li>Dissolve 5 g of mannitol in 250 mL of tap water and dispense into the flask containing the soy flour and agar.</li>
		<li>Add a foam plug and foil to the opening of the flask and autoclave at 121 &#176;C, 15 psi for at least 30 min. 
		CAUTION: This medium easily boils over. Use a flask that can hold at least four times the actual volume of MS agar being autoclaved. A standard pressure cooker can be used in place of an autoclave with similar safety considerations. Alternatively, a microwave oven may be used to sterilize media and materials if access to an autoclave is not possible35,36. High school teachers may also want to collaborate with local colleges or universities to access autoclaved supplies.</li>
		<li>Cool media until comfortable enough to handle. Be careful to not cool it for too long because agar solidifies at temperatures below 50 &#176;C.</li>
		<li>Pour MS agar from the flask into sterile Petri dishes until the bottom of each Petri dish is approximately half full (approximately 25 mL of media per Petri dish). Allow to solidify before using agar plates. Store agar plates in a plastic bag at room temperature until needed. 
		NOTE: Agar plates may also be stored in a refrigerator, especially if antibiotics are needed in the media.</li>
	</ol>
	</li>
</ol>
2. Streak Streptomyces onto Plates for Propagation
<ol>
	<li>Streak Streptomyces strains onto MS agar plates to single colonies using a standard method, such as the quadrant streak method as shown in Sanders, 201237. 
	NOTE: An inoculating loop or sterile toothpicks may be used. Optional: Other media are commonly used for Streptomyces species, such as R2YE and minimal media14.</li>
	<li>Incubate plates at 30 &#176;C.</li>
	<li>Restreak plates by picking a single colony every 4&#8211;6 days. As colonies age they become prone to random mutation.</li>
</ol>
3. Create Glycerol Mycelial Stocks for Bacterial Storage
NOTE: Mycelial stocks may be created for all Streptomyces strains, including whi and bld strains, and other developmental mutants that are unable to complete sporulation.
<ol>
	<li>Macerate 15&#8211;20 colonies in 200&#8211;500 &#181;L of 20% glycerol using a sterile wooden dowel applicator.</li>
	<li>Transfer the slurry of macerated colonies to a 1.5 mL freezer culture tube, using a micropipette or sterile transfer pipette. 
	NOTE: The basics of micropipette use are covered elsewhere38.</li>
	<li>Add enough 20% glycerol to make the final volume 1 mL and gently mix. Store samples at -80 &#176;C. 
	NOTE: Caution! Use cryogenic safety gloves and a lab coat to protect skin from contact with extreme cold. Alternatively, a -20 &#176;C freezer may be used with potential reduction in long term storage time. Avoid excessive freezing and thawing to extend viability.</li>
</ol>
4. Create Glycerol Spore Stocks for Strains Capable of Sporulation
NOTE: Spore stocks are preferable for long-term viability but are only feasible for strains that are capable of completing sporulation.
<ol>
	<li>Spread 100 &#181;L of macerated material (from step 3.1) on an agar plate to obtain a confluent lawn of growth37. 
	NOTE: Spread plating is shown in Sanders37 (Modification: A turntable is not required.). The plate can be turned with one hand while using the other hand to move the spreader in a gentle back and forth motion across the agar media.
	<ol>
		<li>Pipet 100 &#181;L of macerated mycelium to the center of an MS agar plate.</li>
		<li>Sterilize a glass or metal spreading tool by partially submerging in 70% ethanol and passing the spreader once slowly through a Bunsen burner flame to speed the evaporation of the ethanol. 
		CAUTION: Ethanol is highly flammable. Do not place the flaming spreader into ethanol or allow the flaming drop of ethanol to fall into the ethanol dish. Alternatively, use a disposable sterile cotton swab to spread the bacteria on the plate, which requires no ethanol or flame.</li>
		<li>Turn the MS agar plate with one hand, while using a back and forth motion to inoculate the plate with the sterile spreader.</li>
		<li>Incubate for 5&#8211;7 days at 30 &#176;C until the strain is well-sporulated. 
		NOTE: Incubation times will vary depending upon the species and strain.</li>
	</ol>
	</li>
	<li>Add 2 mL of sterile saline (0.8% NaCl) to the center of a confluent lawn of Streptomyces cells that has undergone sporulation.</li>
	<li>Harvest spores by gently rubbing the surface of the mycelium with a sterile inoculating loop (or a cotton bud), slowly moving towards the edge of the lawn.</li>
	<li>Pipet the saline containing spores into a 15 mL conical tube. Vortex vigorously to break spore chains into individual cells.</li>
	<li>Centrifuge at room temperature for 5 min at approximately 2,500 x g.</li>
	<li>Pipet the supernatant and discard. Disperse the spore pellet by vigorous vortex agitation in the small amount of remaining liquid.</li>
	<li>Resuspend the spores in 1 mL of 20% glycerol by drawing the solution up and down with a pipette.</li>
	<li>Transfer the glycerol spore stock to a 1.5 mL freezer tube. Store at -80 &#176;C. 
	CAUTION: Use cryogenic safety gloves and a lab coat to protect skin from contact with extreme cold. Alternatively, a -20 &#176;C freezer may be used with potential reduction of viability in long term storage time. Avoid excessive freezing and thawing to extend viability.</li>
</ol>
5. Perform Mutagenesis
<ol>
	<li>Perform mutagenesis based on the desired outcomes as referenced in the Introduction and recently reviewed by Baltz, 201639. 
	NOTE: Some phenotyping experiments will not require mutagenesis, such as the intent to identify new species from the environment. Methods of random mutagenesis include the use of a transposon8,9,10,11,12,13, UV radiation, or chemicals14. Site-directed mutagenesis15,16,17,18,19,20 techniques can be used to create specific point mutations, deletions, or other types of mutations.</li>
</ol>
6. Compare and Record the Visual Appearance
<ol>
	<li>Take note of colony morphology for each mutant as compared to the wild-type parent strain after streaking all strains as in step 2. Compare the new isolate to that of a well-studied species, such as S. coelicolor or S. venezuelae, when characterizing new Streptomyces species. Note characteristics (Figure 1) such as basic shape, surface of colony (fuzzy, bald, wrinkled, etc.), opacity, elevation, and pigmentation (distinguish between vegetative mycelium, aerial mycelium, and/or surrounding medium).</li>
	<li>Label an MS agar plate and streak wild-type and mutant strains in wedge patterns (Figure 1). Be careful that no strain touches another strain to avoid cross contamination. Note the date and time that strains are streaked onto the plate. Incubate the plates at 30 &#176;C.</li>
	<li>Place grown Petri dishes on colored or white paper to homogenize the background. Write on the paper the strain name, date, incubation temperature, and time from first plate streaking. Take digital picture(s) of the plate with the latter information written on the paper background so that confusion is minimized. 
	NOTE: The writing can be cropped from the photograph at a later time if it is to be used for figure preparation.</li>
</ol>
7. Perform Phase-Contrast Microscopy
<ol>
	<li>Sterilize tweezers by washing in 70% ethanol and then passing through a Bunsen burner flame to evaporate the ethanol. Allow to cool.</li>
	<li>Sterilize coverslips by washing in 70% ethanol and then allowing them to dry in a sterile Petri dish.</li>
	<li>Prepare a coverslip lift of the bacterial growth to be examined.
	<ol>
		<li>Pick up a sterile coverslip with sterile tweezers and place the coverslip on an MS agar plate where bacterial growth is dense. Press on the back of coverslip gently with tweezers to ensure sufficient transfer of bacterial spores and aerial mycelium.</li>
	</ol>
	</li>
	<li>Pick up the coverslip from the plate and place it so that the side with the cell material is facing toward the surface of a microscope slide, with a 15 &#181;L drop of 50% glycerol prepared in water for mounting. Reduce air bubbles by placing the coverslip at a 45&#176; angle to the slide and let it fall onto the 50% glycerol.</li>
	<li>(Optional) Seal with clear nail polish for preservation of the slide.</li>
	<li>Perform phase-contrast microscopy as described by Frohlich40 at various time intervals during the life cycles. 
	NOTE: Repeated imaging allows for a more complete analysis and the ability to detect delays in development. Independently repeat the observations to ensure accuracy and reproducibility.
	<ol>
		<li>Place the prepared slide on the microscope stage and add a drop of immersion oil to the center of the cover slip. Rotate the 100X phase objective in place and set the condenser turret to the proper &#34;matching&#34; phase setting. Focus the image using only the fine adjustment knob once the objective lens is in contact with the oil.</li>
		<li>Examine several fields of view to discern noticeable and consistent differences between mutant strains and the wild type, such as the ability to form spores, spore size and shape, and overall number of spores (see Figure 2). 
		NOTE: Alternatives to phase-contrast microscopy include differential interference contrast (DIC) microscopy40 and simple staining techniques for use with bright field microscopes41, such as crystal violet staining.</li>
	</ol>
	</li>
</ol>
8. Perform Fluorescence Microscopy
<ol>
	<li>Sterilize tweezers by washing in 70% ethanol and then running through a Bunsen burner flame.</li>
	<li>Pick up a sterile coverslip with sterile tweezers and place on an MS agar plate where bacterial growth is evident. Touch the back of the coverslip gently with the tweezers to ensure sufficient transfer of bacterial spores and aerial filaments.</li>
	<li>Pick up the coverslip from the plate and place it so that the side with the cell material is facing up, on cardstock for ease in manipulation of the coverslip.</li>
	<li>Flood the coverslip with ice-cold methanol (~300 &#181;L for a 20 x 20 mm2 coverslip) and let it completely air dry. 
	CAUTION: Methanol is a mutagen. Avoid direct contact with skin.</li>
	<li>Wash the coverslip three times in Phosphate Buffered Saline (PBS)42 by gently dispensing and removing the solution.</li>
	<li>Add 15 &#181;L of 100 &#956;g/ml Propidium Iodide and/or 10 &#956;g/mL Wheat-Germ-Agglutinin conjugated to fluorescein isothiocyanate (WGA-FITC) made in 50% glycerol to a labeled microscope slide.&#160; 
	NOTE: Propidium iodide stains DNA red to detect chromosomal DNA location while WGA-FITC stains the cell wall green.</li>
	<li>Invert coverslip face down onto the drop of fluorescent stain on the glass slide. (Optionally) seal with clear nail polish for preservation of slide.&#160; As the dyes are light-sensitive, incubate for 15 minutes in a darkened or dimly lit room.</li>
	<li>Observe the slides under a 100X objective lens using a phase-contrast or DIC microscope equipped with epifluorescence excitation cubes for propidium iodide (&#39;Texas Red&#39; Filter Set or 535/617 nm excitation/emission maxima) and FITC (FITC filter set or 490/525 nm excitation/emission maxima).</li>
	<li>Place the slide on the stage and focus using the coarse and fine adjustment knobs.</li>
	<li>Observe the fluorescence as shown elsewhere43.</li>
	<li>Use a free image analysis software package that can help in the identification of spores with lower fluorescence intensity44.</li>
</ol>

The authors have nothing to disclose.

Here we present protocols for beginning Streptomyces researchers to initiate studies by including the steps needed to propagate strains and prepare stocks for long-term storage. We then describe the protocols for visual and microscopic characterization of Streptomyces strains. Some typical initial steps in phenotyping developmental mutants are: 1) visual examination of the mutant colonies compared to wild type colonies on agar medium; 2) phase-contrast microscopy; and 3) fluorescence microscopy of sporogenic aerial hyphae. Based upon the phenotype displayed in these three steps, a variety of techniques may be employed to further discern the phenotype of a particular strain.
Initial phenotyping experiments are commonly used to characterize new species, identify mutants of interest, partially characterize mutants, and begin to discern the typical role of a particular gene based on the phenotype of an identified mutant. The methods described here have already been used in university teaching laboratories to identify and characterize a wide variety of Streptomyces mutants, including those with defects in cell division48,49,50,51,52,53,54, sporulation55,56, aerial mycelium formation57,58, antibiotic production59, second messenger signaling47, and chromosome segregation60. These techniques are the vital first steps to determining the phenotype of mutants in general and reveal a large amount of important information about the roles of genes of interest. The methods may easily be extended to other species of Streptomyces and have already been used to describe strains of S. griseus, S. venezuelae, S. scabies, and many other streptomycetes. The video protocols described here are expected to serve as an important resource for new researchers entering fields of Streptomyces research, such as in the area of drug discovery. This includes the new instructors who are working to combat the antibiotic resistance crisis and educating the countless numbers of new undergraduate student researchers joining the crowdsourcing efforts of the Small World Initiative.
The techniques described here can be easily adapted to college and high school classroom use in addition to research laboratories, using the modifications described in the video and text. Students in a first year microscopy module at a small liberal arts college were able to streak strains, take digital photographs of strains grown on agar media, and perform phase-contrast and fluorescence microscopy, which culminated in the submission of a portfolio of multi-paneled figures at the end of the 3 week module, representing 15 h of in-lab work. Approximately 160 first year students were responsible for the initial phenotyping of 320 novel transposon mutants. Undergraduate research students at three institutions participated in the initial phenotyping of additional mutants and the subsequent characterization of many of the strains. The comprehensive data obtained in a relatively short period of time, illustrate the value of the protocols described here. Hundreds of additional mutants have been stored as glycerol mycelial stocks for future characterization.
Following the initial experiments described here, a variety of methods may be employed to extend the quality of information pertaining to strains of interest. If the mutation is unknown, a genotyping method should be employed to determine the type and/or location of the mutation. For example, random transposon mutagenesis of the wild-type chromosome6,7,8,9,10,11,12,13 results in colonies that should undergo initial phenotypic screens such as those described above. Then the location of the transposon should be identified using a technique such as inverse polymerase chain reaction (iPCR)61. Determining the genotype of newly discovered mutants is an important step following initial characterization.
Some commonly used advanced methods for subsequent phenotyping analysis that may be mentioned in the classroom or explored through further research include green fluorescent protein (GFP) tagging to determine localization patterns for the protein of interest, gene expression analyses such as real time quantitative PCR (qPCR) and global gene expression patterns of wild-type versus mutant via RNA Sequencing (RNA-seq). Phenotyping skills are also required for genetic complementation analysis. In a complementation experiment, the wild-type copy of a gene is introduced into a mutated strain to determine whether the newly added allele can compensate for the loss-of-function of the mutated allele. Comparing the phenotype of the complemented strain to that of both the original mutant and the parental, wild-type strain is required.

Streptomycetes are Gram-positive, filamentous soil bacteria known for their ability to produce a variety of secondary metabolites, including over two-thirds of the commercially available antibiotics, as well as anti-tumor, anti-HIV, and anti-parasitic drugs1. S. coelicolor is the most genetically characterized member of the genus2,3 and is the species used in the methods described here. S. coelicolor has a complex life cycle that begins with germination of a single spore, progressing to an extensive, branching vegetative mycelium that grows into the agar medium. As the life cycle proceeds, aerial filaments are formed that break the surface tension of the substrate mycelium and are finally divided into long chains of cells that are ultimately converted into mature, grey-pigmented spores. Dispersion of these newly formed spores constitutes the beginning of the next life cycle4.
Because of its complex pattern of differentiation, S. coelicolor serves as an excellent model for the study of bacterial development. Historically, mutations result in blocks for two major stages of development and produce distinct visual phenotypes. The bld (bald) mutants are blocked for aerial mycelium formation and result in the lack of a fuzzy aerial mycelium imparting a &#34;bald&#34; colony appearance. Mutants inhibited in spore formation and maturation are referred to as whi (white) mutants because they typically fail to produce wild-type levels of the gray spore pigment and the aerial mycelium remains white. Other interesting mutants are inhibited in antibiotic production, cell division, chromosome segregation, or other important processes4,5.
Despite the discovery of many developmental genes in Streptomyces species, there are many more believed to exist based on the lack of saturation of mutant screens. Our laboratories continue to identify new developmental genes using a mini-transposon system that we have constructed. Novel mutant strains isolated in random mutagenesis experiments with our transposon undergo phenotypic screening to identify the possible role of each new gene discovered6,7. The methods for bacterial phenotyping described here are relevant to Streptomyces mutants isolated by transposon mutagenesis8,9,10,11,12,13 and other random methods, such as chemical and ultra-violet (UV) mutagenesis14, as well as the directed construction of mutations such as gene deletions using recombineering15,16 or CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology17,18,19 and point mutations20.
As antibiotic resistance among pathogens becomes increasingly prevalent, the need for new antibiotics becomes increasingly urgent21,22. The &#34;Small World Initiative&#34; (or SWI) is an effective Science, Technology, Engineering and Mathematics (STEM) learning23 and research strategy24 to combat antibiotic resistance through the crowdsourcing of college students, and more recently high school students. Supported courses work includes identifying new soil microbes that produce novel antibiotics (http://www.smallworldinitiative.org). It is believed that a major source of undiscovered antibiotics will continue to be Streptomyces species found in a wide range of soil and water habitats25,26,27,28,29,31. Recently, our laboratory and the work of others have discovered and characterized signaling genes in Streptomyces species that regulate morphology and development, including antibiotic production7,32,33. Changes in expression of these genes result in a change in the amount and timing of antibiotics produced. Skilled phenotyping of new species and new antibiotic-producing mutants will continue to be important. As new instructors and their students become major contributors to the field of drug discovery, training in bacterial phenotyping is necessary for the success of these novice individuals. Furthermore, these experiments are tractable for high school STEM or university microbiology education laboratories. They represent a demonstration of basic microbial genetic principles in a teaching lab setting.

<table border="0" cellpadding="0" cellspacing="0" width="543">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">50% Glycerol</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:121px;">G5516</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Immersion Oil (Type DF)</td>
			<td>Cragille&#160;</td>
			<td>16482</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lens Paper</td>
			<td>Fisherbrand</td>
			<td>11-995</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Water</td>
			<td>GeneMate</td>
			<td>G-3250-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toothpicks (flat)</td>
			<td>Target</td>
			<td>081-22-1957</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette Tips (10 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette Tips (200 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-200Y</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette Tips (1250 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-1250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wooden Applicators 6&#39;&#39;</td>
			<td>Solon Care</td>
			<td>070809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton Swabs</td>
			<td>Fisherbrand</td>
			<td>23-400-124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soy Flour&#160;</td>
			<td>Bob&#39;s Red Mill&#160;</td>
			<td>1516C164</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>VWR amresco life science&#160;</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.8% NaCl (Saline)</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9000V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.2 mL freezer tube</td>
			<td>NEST</td>
			<td>606101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultra low (-80&#176;C) freezer</td>
			<td>SO-LOW</td>
			<td>U85-18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryo Safety Gloves</td>
			<td>Bel-Art</td>
			<td>H13201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5856-500EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover slips #1.5</td>
			<td>Thomas Scientific</td>
			<td>64-0721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slides</td>
			<td>Carolina</td>
			<td>63-2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclave</td>
			<td>Tuttnauer</td>
			<td>2540E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phase-Contrast Microscope&#160;</td>
			<td>Olympus</td>
			<td>BX40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Carolina Biological</td>
			<td>624504</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bunsen Burner</td>
			<td>Carolina Biological</td>
			<td>706706</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-1L-R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (phosphate buffer saline)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WGA-FITC</td>
			<td>Biotium</td>
			<td>29022-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clear nail polish</td>
			<td>OPI</td>
			<td>22001009000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Image J</td>
			<td>NIH</td>
			<td>Free Software</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mL beaker</td>
			<td>Pyrex USA</td>
			<td>1000-T</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L beaker</td>
			<td>Carolina Biological</td>
			<td>721253</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L flask</td>
			<td>Fisherbrand</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 mL flask</td>
			<td>Pyrex USA</td>
			<td>4980</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mL graduated cylinder</td>
			<td>Carolina Biological</td>
			<td>721788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">500 mL graduated cylinder&#160;</td>
			<td>Carolina Biological</td>
			<td>721792</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>22-271825</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera for Microscope</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrile Examination Gloves (Med)</td>
			<td>Bio Excell</td>
			<td>71011002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex Mixer</td>
			<td>Carolina Biological</td>
			<td>701077</td>
			<td></td>
		</tr>
	</tbody>
</table>

visual and microscopic evaluation of streptomyces developmental mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Initial phenotyping experiments are necessary for characterizing new species and strains and can be used as a complimentary approach to the phylogenetics and DNA-DNA hybridization experiments that are used for characterizing new species. Streptomyces mutants resulting from random mutagenesis methods such as chemical, UV, or transposon mutagenesis are typically identified through direct, visual screens on agar plates. Colonies of Streptomyces are examined for changes in phenotype in comparison to the wild-type, parental strain. For example, a lighter colored aerial mycelium may indicate a lower level of gray pigment caused by a defect in sporulation, or the lack of a fuzzy appearance is indicative of a block in aerial mycelium formation (Figure 1). Many streptomycetes produce pigmented antibiotics in vegetative mycelium or surrounding agar. S. coelicolor produces two pigmented antibiotics as well as two non-pigmented ones. Actinorhodin is a blue-pigmented antibiotic and undecylprodigiosin is a red-pigmented antibiotic45. Strains that have undergone initial visual colony screens are then propagated by streaking for single colonies.
Following visual identification of potentially interesting mutants, strains are subjected to microscopic examination. Phase-contrast microscopy is especially suited to examining Streptomyces mutants for developmental defects, using the wild-type strain as a control (Figure 2). Wild type S. coelicolor colonies typically produce an aerial mycelium by about two days of growth at 30 &#176;C on MS agar, and long chains of spores by three days of growth. The life cycle will progress slightly slower or faster on other types of media. It is imperative that the mutants be analyzed under the same growth conditions as the wild type when drawing conclusions about the developmental defects and delays. Bald mutants may produce spores after prolonged growth on agar media. A class of mutants referred to as white mutants may either be delayed for spore formation, show a reduction in the abundance of spores produced, produce spores with shape and/or size defects, or simply produce lower levels of the mature, gray spore pigment46. Other mutants investigated thus far may be delayed or accelerated in the life cycle progression such as the mutants affected for the accumulation of signaling molecules (e.g., cyclic-di-GMP47).
A simple follow-up to the light microscopy technique described above is fluorescence microscopy using propidium iodide to stain chromosomal DNA nucleoids and fluorescently labeled wheat germ agglutinin to stain the cell wall of sporulation septa. Spores lacking a chromosome will be devoid of the red propidium iodide staining, while spores that contain less DNA than usual may be identified if they appear to have decreased staining (Figure 3). Wheat germ agglutinin may be used to stain the cell wall and differences in cell wall staining patterns (i.e., cell division defects) may be observed. In Figure 4 the wild-type strain of S. venezuelae, a species that is recently being used as another model streptomycete because of its faster life cycle and ability to sporulate in liquid45, has been stained with WGA-FITC to elucidate the ladder-like array of division septa that are typically seen early during sporulation.
The initial phenotyping strategies described here should result in the following: 1) Identification of mutant strains of interest for further study; 2) Acquired knowledge about the newly identified mutant and the potential role of the gene that has been mutated; 3) The formulation of a subsequent series of next experimental steps that can be used to further clarify the role of the gene in question. In the case of a newly identified streptomycete from the environment, the researcher will gain knowledge of the potential new species compared to already characterized species of Streptomyces.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57373/57373fig1.jpg" /> 
Figure 1: Photograph of a representative agar plate demonstrating macroscopic colony phenotypes of S. coelicolor. The agar plate shows the fuzzy, gray visual appearance of the aerial mycelium for wild-type S. coelicolor strain MT1110 (WT) in comparison to various random transposon insertion mutants with developmental phenotypes. Mutants are either lacking an aerial mycelium [bald (bld)] or an aerial mycelium with reduced spore pigmentation [white (whi)]. Transposon mutants were generated using mini-Tn5 for insertion mutagenesis6,7. The strains were grown on MS agar for 5 days at 30 &#176;C. Petri dish diameter, 100 mm.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57373/57373fig2.jpg" /> 
Figure 2: Phase-contrast micrographs showing a representative aerial filament for S. coelicolor white mutant strains containing random transposon insertion mutations. (A) Shown is a representative wild type aerial filament that has undergone synchronous, regularly-spaced cell divisions, producing evenly shaped and sized spores (MT1110). (B-F) The remaining panels show the vastly different microscopic phenotypes of various white mutants that were isolated via random transposon mutagenesis. Panel B shows an aerial filament that has not undergone sporulation, but instead has produced bulging areas within the filament. Long arrows in C, D, and F indicate abnormally large spores that are characteristic of mutants MIC42, MIC43, and TH49. The short arrow in panel D indicates an example of a lysed spore compartment. Panel E shows the partially constricted smaller compartments that are typical of the spore chains produced by mutant TH10. The strains were grown on MS agar for 5 days at 30 &#176;C. The mutants are unrelated to those shown in Figure 1. A wild type spore is approximately 1.1 &#181;m on the long axis. Scale bar =&#160;2 &#181;m. <a href="/files/ftp_upload/57373/57373fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57373/57373fig3v2.jpg" /> 
Figure 3: Representative micrographs showing S. coelicolor mutant chromosome segregation phenotypes. (A) A diagram representation shows the typical wild-type, regularly-spaced staining appearance for a chain of spores (every spore contains chromosomal DNA) compared to the intermittent staining pattern for a mutant that displays a chromosome segregation defect. (B) Each pair of panels shows the same spore chain observed by Differential Interference Contrast (DIC) image on the left and a propidium iodide-stained fluorescence image is shown on the right. The wild-type phenotype (a, b) is compared to two random transposon insertion mutants (c, d and e, f). Arrows indicate spores devoid of DNA. The strains were grown on MS agar for 5 days at 30 &#176;C. The mutants shown are unrelated to those in Figure 1 and Figure 2. Scale bar =&#160;1 &#181;m. <a href="/files/ftp_upload/57373/57373fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57373/57373fig4v3.jpg" /> 
Figure 4: Fluorescence micrograph of S. venezuelae wild-type strain stained with WGA-FITC. (A) A diagram of an aerial filament appears smooth with no indentations and no visible signs of sporulation using phase-contrast microscopy in comparison with the ladder-like array of cell wall staining that indicates an early stage of sporulation has begun in that same filament using WGA-FITC under fluorescence microscopy. (B) Micrographs of wild-type S. venezuelae. (a) Smooth aerial filaments are present among chains of spores. (b) Within the mycelium shown in panel a, one aerial filament at an early stage in development possesses a ladder-like array of cell wall deposition. Arrows indicate the regularly-spaced formation of cross-walls stained by WGA-FITC that develop synchronously within a single aerial filament as it undergoes developmentally-associated sporulation. The strain was grown on MYM(Maltose, Yeast extract, Malt extract) MYM agar for 38 h at 30 &#176;C. Scale bar =&#160;2 &#181;m. <a href="/files/ftp_upload/57373/57373fig4v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visual and Microscopic Evaluation of Streptomyces Developmental Mutants at JoVE.com

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

visual-microscopic-evaluation-streptomyces-developmental

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.6K Views.  Otterbein University. Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Video: Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

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.

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 ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

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 ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.