The authors would like to thank Heather Currey for her initial contribution to the study design, and Dr. Swarup Mitra for his critical review of the manuscript. We would also like to thank Dr. Michael B. Harris for comments, refinements and assistance in producing the demonstration of this methodology. The strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990 and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 5P20GM103395-15. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.

1. Caenorhabditis Sieve Construction and Use
<ol>
	<li>Construction protocol
	<ol>
		<li>Acquire 2 lids from 50 mL conical tubes (Figure 1A).</li>
		<li>Remove the center area inside the inner lip of the lids (when viewed from the bottom, Figure 1B) using a Bunsen burner and a hot metal probe or a soldering iron or stepped drill bit. 
		NOTE: Using heat to cut the plastic lid is preferable to a blade because there is less risk of injury.</li>
		<li>Clean and sand the cut edges and top the surface with a curved file or a rotary grinding tool (e.g., Dremel). See Figure 1C.</li>
		<li>Cut the circle of the monofilament mesh to the appropriate diameter (Figure 1D). For this, trace a lid onto a monofilament nylon mesh sheet and cut just inside the line drawn.</li>
		<li>Cut grooves/slashes into the top surface of the lids to enhance the adhesion of the two lids when glue is applied to the plastic (Figure 1E).</li>
		<li>Clean the lids with ethanol and let them dry.</li>
		<li>Apply cyanoacrylate glue to the top surface of both lids, keeping to the outer edge. 
		NOTE: A little glue goes a long way.</li>
		<li>Lay the monofilament mesh, according to Table 1, on one glued lid (Figure 1F). Place the second lid inverted on top of the mesh; both lids must have their tops together. Press firmly together (Figure 1G). Ensure that the mesh is taut. 
		NOTE: As a safety measure, use tweezers to place the mesh on the lids.</li>
		<li>Once the initial layer of glue has dried, apply a ring of cyanoacrylate glue around the outer gap between the lids. Be generous as this adds integrity and prevents any peeling apart or leaking.</li>
		<li>Label the filter with the mesh pore size of the monofilament mesh. 
		NOTE: Here, we use two mesh pore sizes&#8212;20 &#181;m and 50 &#181;m.</li>
	</ol>
	</li>
	<li>Use protocol
	<ol>
		<li>Pre-wet the sieve.
		<ol>
			<li>Pipette a saline solution, such as M9 [5 g of NaCl, 6 g of Na2HPO4, 3 g of KH2PO4, 1 L of ultrapure H2O, and 1 mL of MgSO4 (1 M)]17, through the center of the sieve until a droplet forms, condenses, and drips off the center of the filter bottom (Figure 2A). Optionally, apply a lint-free wipe (e.g., Kimwipe) to the bottom to shape or spread a moisture droplet on the mesh.</li>
			<li>Place the sieve over a 50 mL conical tube. Label the tube as &#39;Waste tube&#39; (Figure 2B).</li>
		</ol>
		</li>
		<li>Wash a population of C. elegans off an agar plate.
		<ol>
			<li>Wash the plate with the M9 buffer and place the worm-containing medium on the topside of the monofilament mesh 1 mL at a time (Figure 2C). Make sure to operate in the center of the mesh. Wash all worms from the plate. 
			NOTE: Typically, 3 mL of M9 for a 60 mm plate is sufficient.</li>
			<li>To minimize the number of worms lost in pipetting, use a glass Pasteur pipette to move the worms off the plate and onto the mesh center (repeat as needed).</li>
			<li>Rinse the filter with the M9 buffer from the top. Again, make sure to operate in the center of the mesh and rinse all worms in that area. Wash as many times as necessary to ensure that all the bacteria and smaller worms have passed through the mesh.</li>
		</ol>
		</li>
		<li>Harvest the size-matched animals from the sieve.
		<ol>
			<li>Attach a new 50 mL conical tube to the top of the sieve, with the adult worms from step 1.2.2.3 facing the inside of the new collection tube (Figure 2D).</li>
			<li>Remove the first tube (waste tube) and quickly flip over the sieve and new tube to keep the droplet from migrating (Figure 2E). 
			NOTE: If sterility is not required, use a lint-free wipe (e.g., Kimwipe) to wick fluid from bottom of the mesh prior to flipping.</li>
			<li>Rinse the mesh with M9 into the new 50 mL tube from the new topside (Figure 2F). Again, operate in the mesh center and maintain a droplet on the underside of the mesh.</li>
			<li>Allow the worms to settle or gently spin them down (e.g., &#60;16 x g) for approximately 1 min (Figure 2G).</li>
			<li>Aspirate the buffer solution from the pellet of worms, ideally down to &#62;0.5 mL, or remove as much fluid as possible without disturbing the pellet.</li>
			<li>Pipette the worms using a glass Pasteur pipette onto an NGM plate and let them dry. Space multiple droplets out on a new plate so they dry faster (Figure 2H).</li>
		</ol>
		</li>
		<li>Clean the sieve.
		<ol>
			<li>Rinse the sieve gently and thoroughly with ethanol and reverse-osmosis water. Let it dry.</li>
			<li>Store it in a clean container for indefinite future use.</li>
			<li>Discard it when the mesh develops a &#34;sag&#34; appearance.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Validation of Caenorhabditis Sieve Sorting Method
<ol>
	<li>General maintenance
	<ol>
		<li>For all experiments, culture worms on a standard Nematode Growth Media17 (1 L of NGM consists of 2.5 g of peptone, 17 g of agar, 3 g of NaCl, 975 mL of double-distilled water, 1 mL of 5 mg/mL cholesterol, 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 25 mL of 1 M KHPO4, and 0.5 mL of 100 mg/mL streptomycin) at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Experimental treatment administration
	<ol>
		<li>Compare the three treatment groups: pick, fluorodeoxyuridine (FUDR), and Caenorhabditis Sieve.
		<ol>
			<li>For the FUDR treatment group, add 100 mg/mL FUDR to the NGM media to a final concentration of 100 &#956;M to prevent any progeny production, and transfer the worms every other day to a fresh NGM plate to avoid food depletion.</li>
			<li>For the pick group, select and transfer the worms manually using a platinum loop.</li>
			<li>For the Caenorhabditis Sieve treatment group, follow step 1.2 and pipette the worms onto an NGM plate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Caenorhabditis Sieve percentage yield
	<ol>
		<li>To quantify how effective the sieve is at sorting C. elegans, grow age-synchronous N2 animals to day 1 of adulthood at 25 &#176;C (i.e., 48 h after egg laying) and then transfer them by picking them to fresh NGM plates (totaling N = 50 or N = 100 animals per treatment group).</li>
		<li>After 24 h of recovery, transfer the population to new NGM plates with the Caenorhabditis Sieve following the above protocol (see step 1.2) and count the number of successfully transferred animals.</li>
		<li>Calculate the percentage yield as the ratio of the number of animals transferred in comparison to the starting number at the beginning of the transfer multiplied by 100 (%).</li>
	</ol>
	</li>
	<li>Healthspan assays 
	NOTE: Score healthspan parameters of the motility class, the pharyngeal pump rate, and both the anterior and the posterior gentle touch response on days 2, 4, 6, and 8 of adulthood for age-synchronized N2 animals maintained at 25 &#176;C.
	<ol>
		<li>Motility tracking
		<ol>
			<li>Assign motility scores based on a class-based system (classes A, B, and C) following the methods of Herndon et al.18. Compare the effects of the three experimental groups using an ordinal logistic statistics model in statistical analysis software. 
			NOTE: Class A individuals move spontaneously in a normal, sinusoidal pattern. Class B individuals move in markedly non-sinusoidal movements and may require prodding to encourage movement. Class C individuals move their head and/or tail in response to prodding but are unable to move across the agar.</li>
		</ol>
		</li>
		<li>Pharyngeal pump rate
		<ol>
			<li>Count the grinder movement of the animal&#8217;s terminal pharyngeal bulb visually under a stereomicroscope at a 600X final magnification for 1 min.</li>
			<li>Conduct a statistical analysis with a one-way ANOVA with &#945; = 0.05 and Bonferroni post-tests with &#945; = 0.05.</li>
		</ol>
		</li>
		<li>Touch response
		<ol>
			<li>Record a gentle touch response and compare between the three treatment groups. Perform the assays based on the methods described by Calixto et al.19.</li>
			<li>Record the anterior and posterior touch response by gently stroking an eyelash pick perpendicularly across the tail or the head (5x each, alternating head and tail) of the animals.</li>
			<li>Score any movement in the opposite direction of the stroke as 1 point on a scale of 0 to 5 for both the anterior and the posterior response.</li>
			<li>Conduct statistical analysis with a one-way ANOVA with &#945; = 0.05 and Bonferroni post-tests with &#945; = 0.05.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fecundity assay
	<ol>
		<li>To determine the use of the Caenorhabditis Sieve&#39;s impact on reproduction, grow age-synchronous N2 animals to day 2 of adulthood at 25 &#176;C.</li>
		<li>60 h after egg lay, transfer animals to new NGM plates with either a platinum pick or the Caenorhabditis Sieve and give them 4 h to recover (dry the sieved plates for 20&#8211;30 min).</li>
		<li>After recovery, individually plate the animals via an eyelash pick to NGM plates, give them 24 h to lay eggs, and remove the animals. Allow the progeny on each plate to develop under normal conditions at 25 &#176;C for another 24 h. 
		NOTE: An eyelash pick is a human eyelash secured to the end of a Pasteur pipette with nail polish and sterilized with ethanol.</li>
		<li>Count the number of viable F1 generation individuals. Perform a statistical analysis using a T-test with &#945; = 0.05. 
		Note: Viable offspring are considered to be eggs that successfully hatch and begin their development through the regular larval cycles.</li>
	</ol>
	</li>
	<li>Fluorescent reporter stress response assays
	<ol>
		<li>Perform three commonly used fluorescent reporter assays to detect potential markers of stress: a DAF-16::GFP translocation into the nuclei of cells in a strain [TJ356- zIs356 (pDAF-16::DAF-16-GFP;rol-6)]20; an hsp-16.2 expression [TJ375- gpIs1 (hsp-16.2p::GFP)]21; and a sod-3 expression [CF1553-muIs84([pAD76]sod-3p::GFP+rol-6[su1006])]22.</li>
		<li>For each assay, culture age-synchronous animals from the three treatment groups at 20 &#176;C and examine them at day 3 of adulthood: use a negative control (on a daily basis, transfer a group of animals manually with a platinum pick), a positive control (on a daily basis, transfer a group of animals manually with a platinum pick plus an established stressor), and the Caenorhabditis Sieve (pass the animals through the sieve and allow them to recover for 30 min on NGM just before imaging).</li>
		<li>In the DAF-16::GFP assay, heat shock the positive control animals at 37 &#176;C for 30 min before imaging20. For the hsp-16.2 assay, heat shock the positive control animals for 90 min, 20 h before imaging21. For the sod-3 assay, treat the positive control animals with 100 mM paraquat for 4 h before imaging22.</li>
		<li>Harvest the worms immediately with an eyelash pick and mount them to a coverslip with 1 &#956;L of a 36% poloxamer 407 surfactant solution to immobilize the worms.</li>
		<li>Sandwich the mounted worms with another coverslip. Mount the two coverslips to a standard glass microscopy slide and image the worms using an 8X magnification on an inverted fluorescent microscope (for an overall magnification of 80X) and a constant exposure with a FITC filter.</li>
		<li>To detect any differences between the three groups in the DAF-16::GFP assay, categorize the animals based on the location of the DAF-16::GFP reporter (nuclear if the reporter translocated to nuclei, cytosolic if the reporter located in cytosol, and intermediate if the reporter located in both nuclei and cytosol).</li>
		<li>Compare the results using an ordinal logistics statistical model in statistical analysis software. For the hsp-16.2 and the sod-3 expression assays, use a one-way ANOVA with &#945; = 0.05 and Tukey&#39;s post hoc tests with &#945; = 0.05 to compare the total fluorescence of the head region.</li>
	</ol>
	</li>
</ol>

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S., Ito, A., Ishii, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+pretreatment+increases+the+X-radiation+resistance+of+the+nematode+Caenorhabditis+elegans.">Oxidative stress pretreatment increases the X-radiation resistance of the nematode Caenorhabditis elegans.</a> Mutation Research. 426 (1), 31-39 (1999).</li><li>Chung, K., Crane, M. M., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+on-chip+rapid+microscopy,+phenotyping+and+sorting+of+C.+elegans.">Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans.</a> Nature Methods. 5 (7), 637-643 (2008).</li></ol>

The authors have nothing to disclose. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Herein, we introduced the design and use of the accessible, effective Caenorhabditis Sieve as a tool for sorting and maintaining C. elegans. This tool has several advantages to manually picking individual animals, washing populations, chemical treatments (e.g., FUDR), and more expensive methods of segregating animals. First, the Caenorhabditis Sieve efficiently and quickly (less than 20 min) sorts progeny from large mixed populations of animals (Table 2). Also, the use of the tool has no detectable toxic effects on the animals&#39; healthspan (Figure 3, Figure 4, Figure 5, Figure 6), does not induce well-described genetic stress reporters (Figure 7, Figure 8, Figure 9), and reduces the amount of foreign chemical that could influence the applied treatment to the culture plates or any molecular assay thereafter. Its ease of use is a significant advantage; a researcher at any stage in their education can be trained to use it (Figure 1 and Figure 2; Protocol).
The materials required for the fabrication and use of the sieve (e.g., pipettes, buffers) are readily available in standard research laboratories; thus, if broken, the Caenorhabditis Sieve is not expensive to replace or repair. The self-constructed nature of the Caenorhabditis Sieve allows it to be a versatile tool: it can be constructed with mesh sizes appropriate for different C. elegans developmental stages and phenotypes. The Caenorhabditis Sieve may also be used in assays conducted in other nematode species or when isolating small organisms, provided the appropriate mesh size is used upon manufacture.
In addition to the benefits this tool provides for C. elegans population maintenance, the Sieve may be used to clean animals before their use in other experimental applications. For example, with the development of microfluidics to conduct C. elegans research comes the issue of chip use and cleaning. Depending on the amount&#160;of bacteria attached to the animals, special precautions need to be taken so as not to clog the microfluidic chip, rendering it unusable29. Often when C. elegans are transferred to a microfluidic chip, residue from the bacterial lawn is brought with them. This residue can and does clog the microfluidic channels, making the chip malfunction, which then requires cleaning or replacement. The device construct in this protocol offers a method for not only harvesting synchronized animals for microfluidics, but also for cleaning the animals prior to their insertion into a microfluidic device. By removing the debris and bacterial residue, taken up when collecting animals, the microfluidic channels are less prone to malfunction, thus increasing the operational life of individual chips and the subsequent throughput of research being conducted with them.

The nematode worm, Caenorhabditis elegans, is a premier model organism. In addition to the straightforward and controlled nature of their cultivation in the laboratory, their entire genome is sequenced1 and the developmental fate of each cell is known2. Due to these features, C. elegans is a widely used model organism for genetic studies. However, along with these beneficial characteristics come some challenges for researchers. Due to their rapid generation time, C. elegans populations can quickly run out of food and/or become mixed populations with multiple generations and developmental stages present at once. Thus, experiments performed on solid nematode growth media (NGM) require researchers to physically move animals to fresh plates before the bacterial food source depletes and new larvae develop. This can be tedious as a frequent transferring of the animals is required to prevent the experimental populations from becoming mixed with offspring generations. Still, some experiments require both large numbers of animals and extended time points (e.g., DNA or RNA extraction in adulthood). This compounds the challenges of accurately maintaining a synchronized population and transferring large numbers of animals.
Current methods of transferring C. elegans cultured on NGM are picking or washing the animals from plate to plate; chemically treating the animals (e.g., with the DNA replication inhibitor fluorodeoxyuridine or FUDR); or using flow cytometry to sort the animals in multi-well plates. Picking involves the use of a hand tool, made with either a thin platinum wire or an eyelash, to manually transfer individual or multiple animals3,4. This method is accurate but requires both skill and time and is a limitation for studies involving large numbers of animals. Picking may also be physically damaging and stressful to the animals by potentially subjecting individuals to unnatural and inconsistent amounts of disturbance and force. Washing involves rinsing a culture dish with a buffer solution and transferring the solution with the animals via glass Pasteur pipette to a new culture plate. This method is rapid and efficient but is not accurate as multiple generations and developmental stages of animals are transferred in bulk. Chemical treatments, such as FUDR, can be dissolved in the culturing media to prevent the production of offspring through blocking any DNA replication, and thus, the gamete production and egg development. While effective, this method must be applied after developmental maturation as to not disrupt the normal developmental processes, and this means that there is still a requirement to transfer the animals prior to its administration3. This method also influences multiple cellular signaling pathways, resulting in noticeable effects on the animals as they age (e.g., a lifespan extension or an altered proteostasis) depending on the strain of C. elegans used5,6,7,8,9,10. Flow cytometry methods automatically sort and transfer individual C. elegans from one multi-well plate to another11. While this method is very effective and efficient, flow cytometry equipment is prohibitively expensive and inaccessible to many researchers. An alternative to transferring animals is to use mutant models that are temperature sensitive, such as fer-15 and fem-1, which become sterile with temperature adjustment12. While using mutant animals is useful in some situations, these specific strains grow slower than wild-type animals and they rely on an altered genome, serving as poor representatives for aging or healthy worms. In addition, the reliance on a temperature shift to induce sterility also results in the absence of a static environment, and temperature changes have been readily shown to influence gene expressions13,14,15. Research groups have previously published techniques describing the use of a mesh to filter C. elegans by size16. However, we were unable to find previous work testing for any changes in the overall health outcomes that may be associated with the use of such filters.
There is, thus, a need within the C. elegans research community for an affordable, efficient, rapid, and accurate method for transferring large numbers of animals between culture plates. We have developed an improved, accessible piece of equipment (named the Caenorhabditis Sieve) and an associated protocol for its manufacture and operation that meets the needs of the C. elegans research community. Herein, we share the design of the Caenorhabditis Sieve and methods for its use, and we demonstrate that its use does not impact the common health or any stress markers when compared to standard manual picking and a treatment with the commonly-used, fertility-restricting chemical FUDR.

<table><tbody><tr><td>Safety glasses</td><td>Uline</td><td>S-21076</td><td></td></tr><tr><td>Protective heat resistant glove</td><td>Grainger</td><td>Item # 3AT17 Mfr. Model # 3AT17 Catalog Page # 1703</td><td></td></tr><tr><td>50 mL conical tube</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>Synthetic Nylon mesh</td><td>Dynamic&lt;br/&gt; Aqua-Supply Ltd</td><td>NTX20 and NTX50</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Scotch Super Glue Liquid</td><td>SAD114</td><td></td></tr><tr><td>Pliers</td><td>Vampliers</td><td>VMPVT-001-8</td><td></td></tr><tr><td>Dremmel tool with circular file</td><td>Lowe&#039;s</td><td>Item # 525945 Model # 100-LG</td><td></td></tr><tr><td>FUDR</td><td>Sigma</td><td>F0503</td><td></td></tr><tr><td>M9 chemicals ( NaCl, Na2HPO4, KH2PO4, MgSO4)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S7653, RES20908-A7, 1551139, M7506</td><td></td></tr><tr><td>NGM plate chemicals (Bactopeptone, Agar, KH2PO4, K2HPO4, CaCl2,Cholesterol, Streptomycin)</td><td>BD Biosciences (bactopeptone) , Lab express (agar), Sigma ( rest)</td><td>BD bioscience 211677, Lab Express 1001,&amp;nbsp; Sigma 1551139, 1551128, C1016, C8667, S6501</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443</td><td></td></tr><tr><td>Paraquat dichloride hydrate</td><td>Sigma</td><td>36541</td><td></td></tr><tr><td>Inverted fluorescence microscope</td><td>Olympus</td><td>FSX100</td><td></td></tr></tbody></table>

caenorhabditis sieve: a low-tech instrument and methodology for sorting small multicellular organisms

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode research community, there is a need for an affordable and effective way to maintain large, age-matched populations of C. elegans. Here, we present a methodology for mechanically sorting and cleaning C. elegans. Our aim is to provide a cost-effective, efficient, fast, and simple process to obtain animals of uniform sizes and life stages for their use in experiments. This tool, the Caenorhabditis Sieve, uses a custom-built lid system that threads onto common conical lab tubes and sorts C. elegans based on body size. We also demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to another allowing for a rapid sorting, synchronizing, and cleaning without impacting markers of health, including motility and stress-inducible gene reporters. This accessible and innovative tool is a fast, efficient, and non-stressful option for maintaining C. elegans populations.

The Caenorhabditis Sieve consists of 2 screw caps, securing an area of woven nylon monofilament mesh smaller than the body diameter of the desired developmental age, used to extract live populations of organisms using a simple washing technique. It attaches to standard conical tubes and uses the mesh screen to mechanically sort animals by body diameter, leaving the desired animals in the tube ready for further maintenance and experimentation (e.g., transfer or genetic harvest). A gentle manual washing with the Caenorhabditis Sieve is quick, approximately 5 min per 60&#8211;100 mm plate, and the organisms are easily recovered from the mesh.
Percentage yield of animals following Caenorhabditis Sieve use 
To establish the percentage yield of the Caenorhabditis Sieve, devices were tested with both 20 &#956;m and 50 &#956;m pore-size meshes on adult animals. A mean yield of a &#62;90% successful animal transfer was achieved for both mesh sizes tested (Table 1).
Monofilament mesh with different size gaps may be used to separate animals of different life stages. Mesh with 20 &#956;m gaps is appropriate for washing away developing embryos and larval stages smaller than the fourth larval stage, retaining the latter (L4; with an average body diameter of 32 &#956;m) and any animals in later life stages, while mesh with 50 &#956;m gaps will allow all other life stages aside from adults (with an average body diameter of 70 &#956;m) to be washed away (Table 2).
Caenorhabditis Sieve use does not impact healthspan metrics 
Motility: In C. elegans the normal sinusoidal movement (i.e., motility) declines with age18 and is a marker of overall health. To determine if the Caenorhabditis Sieve influenced motility, motility scores were compared for pick, FUDR, and Caenorhabditis Sieve treatment groups on days 2, 4, 6, and 8 of adulthood. All the animals across every group (n = 10/group) exhibited normal and spontaneous movement patterns (Class A) at multiple ages throughout adulthood (days 2, 4, 6, and 8 of adulthood; p &#62; 0.05 for multiple comparisons on all days, Figure 3).
Pharyngeal pump rate: The ability of C. elegans&#39; pharyngeal muscles to pump declines with age and is another biomarker of healthspan23. To determine if the Caenorhabditis Sieve influenced the animals&#39; pharyngeal pump rate, pick, FUDR, and Caenorhabditis Sieve treatment groups were compared on days 2, 4, 6, and 8 of adulthood (n = 8 to 10 per group). There was a significant difference between the animals that underwent the picking and FUDR methods on days 6 (p &#60;0.001) and 8 (p &#60;0.001). Also, there was a significant difference between the sieve and FUDR groups on days 6 (p &#60;0.001) and day 8 of adulthood (p &#60;0.001). However, there was no statistically significant difference between the pick and the Caenorhabditis Sieve groups for any day (p &#62;0.05, Figure 4), indicating that the sieve does not impact this measure of healthspan.
Gentle touch response: Response to mechanical stimulus is a physiological marker to assess aging or general health24,25; thus, the impact of different transfer methods on both the anterior and the posterior gentle touch responses were tested. There was no statistically significant difference between the pick, FUDR, and Caenorhabditis Sieve treatment groups (n = 8/group), either anteriorly or posteriorly, for any day of testing (p &#62;0.4 for all comparisons; Figure 5A and 5B).
Fecundity: To establish whether or not the Caenorhabditis Sieve influenced the amount of viable progeny produced by C. elegans, the individual offspring produced in a 24 h period during day 3 of adulthood was counted and compared (n = 20 to 22 per group). The use of the Caenorhabditis Sieve did not significantly impact the number of progeny produced when compared to a pick treatment group (p = 0.61, Figure 6).
Molecular reporter assays 
DAF-16 nuclear translocation: In C. elegans, the activation of the transcription factor DAF-16 is associated with increased stress resistance26. The nuclear localization of DAF-16 was examined in a transgenic nematode strain TJ356, which expresses DAF-16 fused to a green fluorescent protein (DAF-16::GFP)20. Under normal growth conditions, DAF-16::GFP is localized primarily in the cytosol, but under various stressors (e.g., heat stress), it is rapidly translocated into the nucleus20. To test the impact of sorting with the Caenorhabditis Sieve on DAF-16 translocation, DAF-16::GFP localizations were compared in age-matched day-5 adults in a positive control group (heat stress), a negative control group (manual transfer via pick), and a Caenorhabditis Sieve treatment groups (n = 10/group). The transfer with the Caenorhabditis Sieve did not affect the nuclear translocation of DAF-16::GFP, and showed a similar phenotype to the negative control animals (p &#62;0.05, Figure 7).
hsp-16.2 reporter: Small heat shock proteins like HSP-16.2 are biomarkers of a stress response, and they are highly expressed during an exposure to heat shock or oxidative stress agents21,27. The TJ375 strain has a GFP reporter gene fused with an hsp-16.2 promoter&#160;that is not active under normal conditions21. However, after an exposure to a heat shock, HSP-16.2 protein expression is induced, and the animals display high levels of GFP expression21. To test the involvement of the Caenorhabditis Sieve on an HSP-16.2-mediated stress response, the fluorescence density in the pharynx region (n = 10 animals/group) of age-matched animals was compared in day-5 adults between a positive control group (heat stress), a negative control group (picking), and a Caenorhabditis Sieve treatment group. The transfer with the Caenorhabditis Sieve did not significantly induce the expression of HSP-16.2::GFP (hsp-16.2::gfp) when compared to the negative control (p &#62;0.05, Figure 8).
sod-3 reporter: In C. elegans, the anti-oxidant gene that codes for superoxide dismutase 3 (SOD-3) is up-regulated during oxidative stress28. The C. elegans strain CF1553 expresses green fluorescent protein (GFP)-labeled SOD-3 promoter, whose expression is induced by oxidative stressors, such as paraquat5. To test the involvement of the Caenorhabditis Sieve sorting on the antioxidant response in C. elegans, the fluorescence density in the head region of age-matched day-5 adults was compared between a positive control population (100 &#956;M paraquat treatment), a negative control population (manual picking), and a Caenorhabditis Sieve-transferred population. The transfer with the Caenorhabditis Sieve did not significantly induce the expression of sod-3::gfp when compared to the negative control (p &#62;0.05, Figure 9).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58014/58014fig1.jpg" /> 
Figure 1: Caenorhabditis Sieve construction. The progression of the &#39;do-it-yourself&#39; manufacture of the tool is shown. These panels show (A) two 50 mL conical tube caps (B) whose centers have been removed, (C) cut edges smoothed, and (D - F) who are fitted with monofilament mesh corresponding to the desired life stage of C. elegans (see Protocol for details). (G) The completed sieve is also shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58014/58014fig2.jpg" /> 
Figure 2: Step-by-step image representation of Caenorhabditis Sieve use. (A) The sieve is pre-wet with a drop of M9 solution, and (B) fit on top of a 50 mL conical tube. (C) 1 mL of M9 solution with worms is pipetted on the topside of the sieve, (D) a 50 mL conical tube is placed on top of the sieve with the worms facing the inside of the tube, and (E) the sieve with a newly attached upper tube is quickly flipped over. (G) The sieve is rinsed with M9 carrying the desired animals into the new 50 mL tube and the worms are allowed to settle by gravity to the bottom of the tube. (H) The worms are pipetted and placed as droplets on a fresh NGM plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig2large.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/58014/58014fig3.jpg" /> 
Figure 3: Caenorhabditis Sieve did not impact motility throughout lifespan. This figure shows the motility class distribution of animals at days 2, 4, 6, and 8 of adulthood for pick, FUDR, and Caenorhabditis Sieve treatment groups. Class A animals moved normally and spontaneously, class B animals moved abnormally and may have required prodding, and class C animals were unable to move. There was no difference between Caenorhabditis Sieve, pick and FUDR groups (p &#62;0.05). Three replicates (n = 10 animals per treatment plate) were conducted and analyzed with Ordinal logistic model. class B animals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig3large.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/58014/58014fig4.jpg" /> 
Figure 4: Caenorhabditis Sieve use did not impact pharyngeal pumping throughout the lifespan. This figure shows the pharyngeal pump rates of pick, FUDR, and Caenorhabditis Sieve treatment groups, compared on days 2, 4, 6, and 8 of adulthood. The asterisks denote a significance between the pick, sieve, and FUDR treatment groups for the days specified (*** p &#60;0.05). There was no difference between the Caenorhabditis Sieve and the pick group (p &#62; 0.05). Two replicates (N = 10 animals per treatment plate) were conducted and analyzed with a one-way ANOVA and a Bonferroni post-test. The bars represent the mean &#177; the standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58014/58014fig5.jpg" /> 
Figure 5: Caenorhabditis Sieve use did not impact anterior touch response throughout lifespan. These panels show (A) the anterior and (B) the posterior touch response count of pick, FUDR, and Caenorhabditis Sieve treatment groups compared on days 2, 4, 6, and 8 of adulthood. Two replicates were conducted with N = 10 for each treatment group and compared with a one-way ANOVA and a Bonferroni post-test (p = 0.4 and p = 0.9 for anterior and posterior, respectively). The bars represent the mean &#177; the standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58014/58014fig6.jpg" /> 
Figure 6: Caenorhabditis Sieve did not impact the amount of viable progeny on day 3 of adulthood. This figure shows the viable progeny of day-3 adults after a 24 h egg-laying period, spanning day 3 of adulthood for parent animals. The bars represent the mean &#177; the standard error of the mean. N = 20 - 22 animals from at least two separate biological replicates per treatment group. The treatment groups are compared with a t-test, (p &#62;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58014/58014fig7.jpg" /> 
Figure 7: Caenorhabditis Sieve did not affect nuclear translocation of DAF-16::GFP. These panels show representative images of the DAF-16 translocation of (A) a heat shock group (the positive control), (B) a pick group (the negative control), and (C) a Caenorhabditis Sieve treatment group. (D) The animals in the positive control group displayed an activation of the DAF-16 nuclear translocation. The Caenorhabditis Sieve did not induce a nuclear translocation and displayed cytosolic fusion protein similar to the animals in the negative control group. N = 10 animals per treatment group from at least three separate biological replicates. The treatment groups were compared with a one-way ANOVA and a Tukey&#39;s post hoc test. Scale bar = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58014/58014fig8.jpg" /> 
Figure 8: Caenorhabditis Sieve did not affect the expression of hsp16.2::gfp. The HSP-16.2 expressions (in arbitrary fluorescence units) of a heat shock group (the positive control), a pick group (the negative control), and a Caenorhabditis Sieve treatment group are compared. The asterisks denote a high expression of hsp16.2::gfp for the positive control group which was significantly different from the other treatment groups (*** p &#60;0.05). The Caenorhabditis Sieve did not affect the hsp16.2::gfp expression and displayed fluorescence intensities similar to animals in the negative control group (p &#62;0.05). Three replicates were conducted with N = 10 for each treatment group and compared with a one-way ANOVA and a Tukey&#39;s post hoc test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58014/58014fig9.jpg" /> 
Figure 9: Caenorhabditis Sieve did not affect the expression of sod-3::gfp. The SOD-3 expressions (in arbitrary fluorescence units) of a 100 &#956;M paraquat group (the positive control), a pick group (the negative control), and a Caenorhabditis Sieve treatment group are shown. The asterisks denote a high expression of sod-3::gfp for the positive control group which was significantly different from the other groups (*** p &#60;0.05). The Caenorhabditis Sieve did not affect the sod-3::gfp expression and displayed fluorescence intensities similar to the animals in the negative control group (p &#62;0.05). Three replicates were conducted with N = 10 for each treatment group and compared with a one-way ANOVA and a Tukey&#39;s post hoc test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58014/58014fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mesh size</td>
			<td>N = 50</td>
			<td>N = 100</td>
		</tr>
		<tr>
			<td>20 &#956;m</td>
			<td>95.33%</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#956;m</td>
			<td>99.00%</td>
			<td>93.33%</td>
		</tr>
	</tbody>
</table>
Table 1: Percentage yield of mesh sizes. This table shows the results of a 20 &#956;m device tested with N = 50 adults for 3 replicates and a 50 &#956;m device tested with N = 50 adults for 2 replicates and with N = 100 for 3 replicates.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mesh Size</td>
			<td>Developmental Stage</td>
			<td>Body Diameter</td>
		</tr>
		<tr>
			<td>20 &#956;m</td>
			<td>Larval Stage 4</td>
			<td>~35 &#956;m</td>
		</tr>
		<tr>
			<td>50 &#956;m</td>
			<td>Day 1 Adult</td>
			<td>~70 &#956;m</td>
		</tr>
	</tbody>
</table>
Table 2: Mean body diameter. This table displays the mean body diameter acquired by averaging 3 measurements equally spread across each worm for 15 animals at each measured life stage.

Watch this Scientific Journal Video about Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms at JoVE.com

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

The current protocol includes a methodology for the sorting and cleaning of age-matched populations of Caenorhabditis elegans. It uses a simple, inexpensive, and efficient custom-made tool to obtain a large experimental population of nematodes for research.

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode ...

caenorhabditis-sieve-low-tech-instrument-methodology-for-sorting

Nanyang Technological University

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6.2K Views.  University of Alaska Fairbanks. Het huidige protocol bevat een methode voor het sorteren en het schoonmaken van leeftijd-matched populaties van Caenorhabditis elegans. Het gebruikt een eenvoudig, goedkoop, en efficiënt instrument is op maat te verkrijgen een grote experimentele populatie van nematoden voor onderzoek.

Video: Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

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The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

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The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

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Environment

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Genetics

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Immunology and Infection

Automatic Image Processing to Determine the Community Size Structure of Riverine Macroinvertebrates

Body size is an important functional trait that can be used as a bioindicator to assess the impacts of perturbations in natural communities. Community size structure responds to biotic and abiotic gradients, including anthropogenic perturbations across taxa and ecosystems. However, the manual measurement of small-bodied organisms such as benthic macroinvertebrates (e.g., &gt;500 &#181;m to a few centimeters long) is time-consuming. To expedite the estimation of community size structure, here, we developed a protocol to semi-automatically measure the individual body size of preserved river macroinvertebrates, which are one of the most commonly used bioindicators for assessing the ecological status of freshwater ecosystems. This protocol is adapted from an existing methodology developed to scan marine mesozooplankton with a scanning system designed for water samples. The protocol consists of three main steps: (1) scanning subsamples (fine and coarse sample size fractions) of river macroinvertebrates and processing the digitized images to individualize each detected object in each image; (2) creating, evaluating, and validating a learning set through artificial intelligence to semi-automatically separate the individual images of macroinvertebrates from detritus and artifacts in the scanned samples; and (3) depicting the size structure of the macroinvertebrate communities. In addition to the protocol, this work includes the calibration results and enumerates several challenges and recommendations to adapt the procedure to macroinvertebrate samples and to consider for further improvements. Overall, the results support the use of the presented scanning system for the automatic body size measurement of river macroinvertebrates and suggest that the depiction of their size spectrum is a valuable tool for the rapid bioassessment of freshwater ecosystems.

The article is based on the creation of an adapted protocol to scan, detect, sort, and identify digitized objects corresponding to benthic river macroinvertebrates using a semi-automatic imaging procedure. This procedure allows the acquisition of the individual size distributions and size metrics of a macroinvertebrate community in about 1 h.

Body size is an important functional trait that can be used as a bioindicator to assess the impacts of perturbations in natural communities. Community ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Bioengineering

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Biology

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

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