The authors would like to acknowledge Suzanne Howard and Justin Finne. This work was funded by the Wellesley College Neuroscience Department. N2 worms were provided by the CGC which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).&#160;The publication fees for this article were supported by the Wellesley College Library and Technology Services Open Access Fund.

1. Prepare the micro-incinerator
<ol>
	<li>Attach a loop holder accessory guide to the micro-incinerator by clipping it onto the outer barrel.</li>
	<li>Plug in micro-incinerator to a standard 120 V or 230 V electrical outlet as appropriate. 
	NOTE: This can be done on a benchtop or in a laminar flow hood.</li>
	<li>Turn the micro-incinerator to the high setting and allow it to warm up for 10-20 min depending on the manufacturer&#39;s instructions to reach an optimum temperature of 800-825 oC. 
	&#8203;NOTE: If keeping the incinerator on for longer than 3 h, the lower temperature setting (500 oC) can be used as a standby setting. According to manufacturers&#39; user manuals, this extends the usable life of the equipment.</li>
</ol>
2. Sterilize the pick, spatula, or scalpel
<ol>
	<li>Insert the instrument into the cylindrical sterilization area without touching the sides by sliding along the guide21. 
	NOTE: If sterilizing a scalpel, it is critical to avoid touching the ceramic wall with the blade. Scraping the ceramic wall may compromise the integrity of the heating unit.</li>
	<li>Hold the instrument in the sterilization area for 5-7 s.</li>
	<li>Remove the instrument without touching the sides by sliding backward along the guide.</li>
	<li>For picks, allow the instrument to cool for 3-5 s before touching a worm to avoid burning it. 
	NOTE: Scalpels or spatulas that are not allowed to cool will singe the agar.</li>
	<li>After picking worms, re-insert the pick in the chamber for 5-7 s to incinerate any worms on the pick.</li>
</ol>
3. Comparative method - sterilization of instruments using a Bunsen burner
<ol>
	<li>Connect Bunsen burner to the gas line using rubber tubing. Make sure to secure the tubing tightly and position the burner away from overhead objects.</li>
	<li>Turn on gas by turning the knob on the gas line.</li>
	<li>Ignite the burner using a striker or lighter.</li>
	<li>Adjust flame using the gas knob and the air intake until a blue cone is visible.</li>
	<li>Hold the pick, spatula, or scalpel in the flame until it glows red.</li>
	<li>For picks, allow the instrument to cool for 3-5 seconds before touching a worm to avoid burning it. 
	NOTE: Scalpels or spatulas that are not allowed to cool will singe the agar.</li>
	<li>After picking worms, re-insert the pick in the flame to incinerate any worms on the pick.</li>
</ol>
4. Experiment
<ol>
	<li>Culture OP50 Escherichia coli (E. coli) bacteria in Luria Broth22 overnight on a 37 oC shaker.</li>
	<li>After overnight culture, dilute the culture in sterile water at a ratio of 1:100. 
	NOTE: This dilution factor was chosen to ensure the separation of colonies after plating.</li>
	<li>Dip a worm pick sterilized using a Bunsen burner in the bacterial solution, re-sterilize with the Bunsen burner, and swirl it in 100 mL of sterile water.</li>
	<li>Plate the 100 &#181;L of water on a 10 cm LB agar22,23 Petri dish, spread the water using a sterile cell spreader, and incubate at room temperature for 24 h with the lid on.</li>
	<li>Perform steps 4.3-4.4 using a worm pick sterilized using a micro-incinerator.</li>
	<li>As a positive control, perform steps 4.3-4.4 without re-sterilization.</li>
	<li>As a negative control, perform steps 4.3-4.4 using sterile water in place of the bacterial solution.</li>
	<li>Count the colonies manually after the incubation period.</li>
</ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inquiry-based+approach+to+study+the+synapse:+Student-driven+experiments+using+C.+elegans.">An inquiry-based approach to study the synapse: Student-driven experiments using C. elegans.</a> Journal of Undergraduate Neuroscience Education: JUNE: A Publication of FUN, Faculty for Undergraduate Neuroscience. 15 (1), 44-55 (2016).</li><li>Raley-Susman, K. M., Gray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+gerontogenes+in+the+nervous+system:+a+multi-level+neurogenomics+laboratory+module+for+an+intermediate+neuroscience+and+behavior+course.">Exploration of gerontogenes in the nervous system: a multi-level neurogenomics laboratory module for an intermediate neuroscience and behavior course.</a> Journal of Undergraduate Neuroscience Education. 8 (2), 108-115 (2010).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: The Online Review of C. Elegans Biology. , 1-11 (2006).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments: JoVE. (64), e4019 (2012).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+I:+yeast,+Drosophilia,+and+C.+Elegant.+C.+Elegans+Maintenace.">Biology I: yeast, Drosophilia, and C. Elegant. C. Elegans Maintenace.</a> JoVE Science Education Database. , (2022).</li><li>Hope, I. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It+says+in+the+books+that+ethanol+burns+with+a+cool+flame.">It says in the books that ethanol burns with a cool flame.</a> Journal of Chemical Education. 77 (11), 1488 (2000).</li><li>Mojtabai, F., Kaufman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Learning by accident. V. 2. , (2005).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Laboratory+Techniques.+Introduction+to+the+Bunsen+Burner.">General Laboratory Techniques. Introduction to the Bunsen Burner.</a> JoVE Science Education Database. , (2022).</li><li>Bykowski, T., Stevenson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique.">Aseptic Technique.</a> Current Protocols in Microbiology. 56 (1), 98 (2020).</li><li>Katz, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+streak+plate+protocol.">The streak plate protocol.</a> American Society for Microbiology Laboratory Protocols. , (2008).</li><li>Public Health Agency of Canada. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agency+of+Canada+Canadian+Biosafety+Handbook.">Agency of Canada Canadian Biosafety Handbook.</a> Government of Canada. , (2016).</li><li>Emmert, E. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASM+Task+Committee+on+Laboratory+Biosafety+Biosafety+guidelines+for+handling+microorganisms+in+the+teaching+laboratory:+development+and+rationale.">ASM Task Committee on Laboratory Biosafety Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale.</a> Journal of Microbiology &amp; Biology Education. 14 (1), 78-83 (2013).</li><li>Collins, C. H., Pal, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+Hazards+in+Microbiology.">Health Hazards in Microbiology.</a> Handbook of Laboratory Health and Safety Measures. , (1990).</li><li>Laboratory Biosafety Manual. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/I/item/9789240011311">https://www.who.int/publications/I/item/9789240011311</a> (2020)</li><li>Fleming, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Safety+Principles+and+Practices.">Laboratory Safety Principles and Practices.</a> American Society for Microbiology. , (1995).</li><li>Gordon, R. C., Davenport, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+modification+to+improve+usefulness+of+the+Bacti-Cinerator.">Simple modification to improve usefulness of the Bacti-Cinerator.</a> Applied Microbiology. 26 (3), 423 (1973).</li><li>Cold Spring Harbor. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LB+(Luria-Bertani)+liquid+medium.">LB (Luria-Bertani) liquid medium.</a> Cold Spring Harbor Protocols. 2006 (1), (2006).</li><li>Cold Spring Harbor. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media+containing+agar+or+agarose.">Media containing agar or agarose.</a> Cold Spring Harbor Protocols. 2006 (1), (2006).</li></ol>

No conflicts of interest were disclosed.

C. elegans is a model organism well suited for exercises in undergraduate teaching laboratories. The use of micro-incinerators in place of open flames provides benefits in both research laboratories and classroom laboratories. In fact, undergraduate laboratory courses may pose a higher risk of accidental fires given the number of newly trained scientists in the room. In addition, the risk of fire increases when ethanol is used to sterilize instruments near the source of flame as ethanol vapors are ignitable. The portability also confers an advantage for classrooms where gas lines are not installed at each bench. This method has been employed in teaching and research laboratories at our institution, resulting in no increases in contamination and zero laboratory safety accidents since its incorporation in 2016. 
To ensure compatibility with micro-incinerators, a range of picks with different mountings, wire compositions, and wire gauges were tested. Wire composition included 100% platinum as well as 90% platinum/10% iridium with a wire thickness of 30-32 G, and regardless of thickness and composition, the heating method did not compromise wire integrity. Mountings included two different types of commercially available pick handles and in-house-made glass mountings from Pasteur pipettes. Note that picks do not glow red-hot as they do in the flame. However, sufficient sterilization is still achieved as long as the sterilizer has reached the proper temperature. Thus, it is critical to allow the micro-incinerator to warm up for 10 or 20 min, as indicated in the manufacturer's instructions. Keeping the instrument in the chamber for at least 5 s to achieve sterilization is also critical. Leaving the instrument in the chamber longer than 7 s will not cause harm to the instrument but is unnecessary. While this is a relatively straightforward procedure with minimal steps and is unlikely to need troubleshooting, it may take some practice to learn to steady the instrument in the barrel without touching the sides.
In order to replace an open flame, a sterilization method must cover all of the applications used in a laboratory. In addition to sterilizing instruments, C. elegans laboratories may also use Bunsen burners to create a sterile field to do other tasks such as pouring plates or inoculating cultures13. However, whether this creates a sterile field or draws in still-viable contaminants remains controversial14. Although not an option at all institutions, a biosafety cabinet or laminar flow hood can be used for these purposes, allowing a laboratory to function without the use of open flames. Instruction manuals for most micro-incinerators recommend against use for sterilization of scalpel blades because scraping of the inner walls damages the sterilizer. However, if carefully using a guide, one can sterilize a scalpel blade or spatula without touching the inner walls. This extends the use of the technique to allow for chunking without using a flame and allows a C. elegans laboratory to function without switching techniques between sterilizing different objects.
As outlined in Table 1, micro-incinerators offer increased safety, increased compatibility with laminar flow hoods, and increased portability over flame-based methods but have limitations. They are more costly than the other methods and require a warm-up time before sterilization temperatures are achieved. In conclusion, this method which is routinely used in many microbiology laboratories, may be applicable in some C. elegans research and teaching laboratories seeking to improve laboratory safety without compromising sterility.

The model organism C. elegans is well-suited for both research and education at primarily undergraduate institutions (PUIs) due to the low cost, ease of maintenance, and range of applications1,2,3,4. In order to handle worms - for example, to move a worm from one plate to another, experimenters can use a worm pick. A variety of picks can be made or purchased for use with C. elegans. Picks are most commonly made using a platinum or platinum/iridium tip, mounted in a glass, metal, or wooden handle. Glass handles can be made in-house by melting a Pasteur pipette around a platinum wire until the wire is secure. Additional information on C. elegans husbandry, including how to grow and maintain worms and their food sources, can be found in WormBook5 and other sources6,7,8.
When working with C. elegans, aseptic techniques are typically used to prevent contamination with microbes and fungi. Examples of aseptic techniques include sterilization of instruments, autoclaving of reagents, and conducting work in sterile fields. Worm picks are typically sterilized using an open flame9. Additionally, sterilization of the worm pick incinerates worms, thus preventing accidental mix-ups of strains when working with multiple worm strains. Typical methods for sterilizing worm picks involve an open flame from either a Bunsen burner, ethanol lantern, or standard lighter (Table 1). We were motivated to seek safer alternatives to existing methods in the laboratory when an undergraduate student unknowingly spilled ethanol while filling an ethanol lantern and accidentally started a small fire when igniting the lantern. Unfortunately, many accidents have been reported using ethanol lanterns10,11,12. Fortunately, alternative sterilization methods have been validated for use in microbiology, and the purpose of this article is to demonstrate how to use this equipment to sterilize instruments for use with C. elegans.
In microbiology laboratories, the aseptic technique is also critical. Serological loops and wires made of platinum are sterilized either using an open flame13 or a micro-incinerator14,15,16. Other names for micro-incinerators include micro-sterilizers or bacto-incinerators. Advantages of the micro-incinerator over traditional flame methods include reduced fire hazard, elimination of spattering of incinerated materials, and ability to work in a laminar flow hood/biosafety cabinet16,17,18. In fact, both the American Society of Microbiology and the World Health Organization recommends using micro-incinerators over the use of an open flame17,19,20. In comparison with Bunsen burners, micro-incinerators also do not require a gas line, which some laboratories might not have, or might not have located at each bench for students to use. Inspired by these advantages, a protocol was developed to replace the use of flame with micro-incinerators for sterilization of commonly used instruments such as picks, spatulas, and scalpels in the C. elegans laboratory. This method may be appropriate for instructors and researchers seeking to enhance safety and/or flexibility when working with C. elegans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>90% platinum 10% iridium wire</td>
			<td>Tritech</td>
			<td>PT-9010</td>
			<td>Other sources and wire compositions may be used.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A6013</td>
			<td>LB agar ingredient</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>50-110-1225</td>
			<td>Other Bunsen burners may be used</td>
		</tr>
		<tr>
			<td>Ethanol Lamp</td>
			<td>Carolina</td>
			<td>706604</td>
			<td>Included here as a reference to Table 1</td>
		</tr>
		<tr>
			<td>Lighter</td>
			<td>Carolina</td>
			<td>706636</td>
			<td>Included here as a reference to Table 1</td>
		</tr>
		<tr>
			<td>Loop holder accessory</td>
			<td>Fisher</td>
			<td>22-630-002</td>
			<td>Referred to in the manuscript as &#34;guide&#34;</td>
		</tr>
		<tr>
			<td>Micro-incinerator</td>
			<td>Thomas Scientific</td>
			<td>1154J15</td>
			<td>There are many companies that sell similar equipment. Similar models also sold by Benchmark Scientific (B1001),&#160; Fisher Scientific (22-630-001), Carolina (703400), and BT Lab Systems (BT1702).</td>
		</tr>
		<tr>
			<td>N2 worms</td>
			<td>CGC</td>
			<td>N2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td>LB ingredient</td>
		</tr>
		<tr>
			<td>OP50 E. coli</td>
			<td>CGC</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher</td>
			<td>08-772B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pick handle</td>
			<td>Tritech</td>
			<td>TWPH1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Fisher</td>
			<td>12-000-161</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fisher</td>
			<td>12-000-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Fisher</td>
			<td>14-374</td>
			<td>Other spatulas will work</td>
		</tr>
		<tr>
			<td>Sterile cell spreaders</td>
			<td>VWR</td>
			<td>76206-438</td>
			<td>Other cell spreaders may be used as long as they are sterile</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma Aldrich</td>
			<td>T7293</td>
			<td>LB ingredient</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y1625</td>
			<td>LB ingredient</td>
		</tr>
	</tbody>
</table>

a flame-free method for sterilizing c. elegans picks, spatulas, and scalpels

Caenorhabtidis elegans (C. elegans) is an optimal model organism for research and education at primarily undergraduate institutions. Undergraduates can quickly learn the sterile technique required to maintain C. elegans cultures. Sterilization of platinum picks used to transfer worms from one plate to another is traditionally done by holding the pick in a flame from a Bunsen burner or ethanol lantern. However, Bunsen burners require a gas source, and both pieces of equipment pose the risk of accidental fire associated with an open flame. Demonstrated here is a technique for sterilizing worm picks, spatulas, and scalpels using an infrared bacteriological loop micro-incinerator. This equipment requires only an electrical outlet and minimizes potential fire hazards. By lowering risk and gas requirements, this technique is well suited for research and teaching in an undergraduate setting.

A simple experiment (section 4) was devised to demonstrate the relative contamination rates using a micro-incinerator versus a flame (Figure 1, Table 2). While these results represent contamination rates across methods, it would not be necessary to repeat this method to use the micro-incinerator technique. The experiment was run in triplicate. Negative control was sterile water with no bacteria. The positive control used neither sterilization technique: the pick was dipped in the diluted OP50 culture then transferred directly to the sterile water. All replicates and conditions were run in parallel on the same day. In all three negative control plates, zero colonies were counted. A mean count of 4.7 (&#177; 0.3 SEM) colonies was obtained when the Bunsen burner was used to sterilize picks. A mean count of 3.3 (&#177;1.2 SEM) colonies was observed in the micro-incinerator condition. However, a mean count of 298.3 (&#177;17.9 SEM) colonies was obtained in the positive control condition. A 2-tailed t-test assuming equal variance comparing the Bunsen burner to micro-incinerator yielded no statistically significant difference, p = 0.35. Thus, the micro-incinerator achieved sterility results equally effective to the Bunsen burner.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63578/63578fig01.jpg" /> 
Figure 1: Bacterial counts across sterilization methods.&#160;Picks were dipped in 1:100 OP50 culture in sterile water and then sterilized using a Bunsen burner or micro-incinerator. Picks were then dipped in sterile water, plated on LB agar22,23, and incubated for 24 h at room temperature, and colonies were counted manually. Positive control had no sterilization, and negative control used sterile water with no bacteria. n = 3 replicates per condition. Note: y-axis is broken to allow separation of data points in relevant portions of the graph. <a href="https://www.jove.com/files/ftp_upload/63578/63578fig01large.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>Sterilization Method</td>
			<td>Cost</td>
			<td>Lab Requirements</td>
			<td colspan="2">Advantages</td>
			<td colspan="3">Disadvantages</td>
		</tr>
		<tr>
			<td>Micro-incinerator</td>
			<td>$365&#8211;530</td>
			<td>120 V or 230 V outlet</td>
			<td colspan="2">&#8226; Portable 
			&#8226; No open flame or exposed heating element 
			&#8226; Useable in laminar flow hoods and biological safety cabinets</td>
			<td colspan="3">&#8226; Warm up time 
			&#8226; Higher cost</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>$24&#8211;169</td>
			<td>Gas Line</td>
			<td colspan="2">&#8226; Fast set up 
			&#8226; Low cost</td>
			<td colspan="3">&#8226; Open Flame 
			&#8226; Not recommended for use in laminar flow hood or biological safety cabinet</td>
		</tr>
		<tr>
			<td>Ethanol Lamp</td>
			<td>$11</td>
			<td>None</td>
			<td colspan="2">&#8226; Fast set up 
			&#8226; Low cost 
			&#8226; Refillable</td>
			<td colspan="3">&#8226; Open Flame 
			&#8226; Safety hazard when refilling 
			&#8226; Not recommended for use in laminar flow hood or biological safety cabinet 
			&#8226; Not allowed at certain institutions</td>
		</tr>
		<tr>
			<td>Lighter</td>
			<td>$5&#8211;8</td>
			<td>None</td>
			<td colspan="2">&#8226; Low cost 
			&#8226; Accessible 
			&#8226; Disposable 
			&#8226; Refillable</td>
			<td colspan="3">&#8226; Open Flame 
			&#8226; Hand operated 
			&#8226; Not recommended for use in laminar flow hood or biological safety cabinet</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of methods for sterilizing instruments. Four methods for sterilizing instruments were compared based on advantages, disadvantages, costs, and laboratory requirements.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Replicate</td>
			<td colspan="4">Condition</td>
		</tr>
		<tr>
			<td>Micro-incinerator</td>
			<td>Bunsen Burner</td>
			<td>Negative Control</td>
			<td>Positve Control</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1</td>
			<td>5</td>
			<td>0</td>
			<td>278</td>
		</tr>
		<tr>
			<td>2</td>
			<td>4</td>
			<td>4</td>
			<td>0</td>
			<td>334</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5</td>
			<td>5</td>
			<td>0</td>
			<td>283</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>3.3</td>
			<td>4.7</td>
			<td>0.0</td>
			<td>298.3</td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>1.2</td>
			<td>0.3</td>
			<td>0.0</td>
			<td>17.9</td>
		</tr>
	</tbody>
</table>
Table 2: Raw data of bacterial counts across sterilization methods.&#160;Picks were dipped in 1:100 OP50 culture in sterile water and then sterilized using a Bunsen burner or micro-incinerator. Picks were then dipped in sterile water, plated on LB agar22,23, and incubated for 24 h at room temperature, and colonies were counted manually. Positive control had no sterilization, and negative control used sterile water with no bacteria. n = 3 replicates per condition. Mean and SEM reported below individual counts.

Watch this Scientific Journal Video about A Flame-Free Method for Sterilizing C. elegans Picks, Spatulas, and Scalpels at JoVE.com

A Flame-Free Method for Sterilizing C. elegans Picks, Spatulas, and Scalpels

This article describes a method for sterilizing worm picks, spatulas, and scalpels using a micro-incinerator in place of an open flame.

A Flame-Free Method for Sterilizing C. elegans Picks, Spatulas, and Scalpels

Caenorhabtidis elegans (C. elegans) is an optimal model organism for research and education at primarily undergraduate institutions. Undergraduates can ...

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4.5K Views.  Wellesley College. This article describes a method for sterilizing worm picks, spatulas, and scalpels using a micro-incinerator in place of an open flame.

Video: A Flame-Free Method for Sterilizing C. elegans Picks, Spatulas, and Scalpels

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...