We acknowledge Noah Joseph (Northeastern Bioengineering Department) for the copper plate machining.

1. Manufacturing and preparing each component of the cooling stage
NOTE: The cooling stage comprises several components (see Table of Materials). Most of the components are off-the-shelf. The sapphire window requires a custom order, while the copper plate, holding bracket, and isolation plate can be manufactured on-site with a computer numerical control mill or 3D printer. After the initial manufacturing, the later assembly process takes around 2-3 h.
<ol>
	<li>Use a computer numerical control mill to machine the copper plate from a 170 mm x 120 mm x 3 mm, 99.9% pure copper metal sheet (Figure 2A). The 2D drawing for this manufacturing is provided in the Supplementary File 1. Use fine-grit sandpaper to remove any sharp edges and dirty residue.</li>
	<li>To manufacture the holding bracket and the isolation plate, use a 3D printer and 1.75 mm diameter polylactic acid (PLA) filament (Figure 2B,C). For better quality, the 3D printer should provide a layer height finer than 0.2 mm. 3D models are provided in Supplementary File 2 and Supplementary File 3.</li>
</ol>
2. Constructing the water-cooling assembly
<ol>
	<li>Prepare the platinum-cured silicone tube, pump tank, copper cooling block, and radiator (Figure 3A) for building the water-cooling assembly. Prepare a razor blade, scissors, and hex key ready for use. Be aware of electrical hazards due to the use of water throughout assembly.</li>
	<li>Cut the silicone tube into three sections with suggested lengths of 40 cm, 50 cm, and 80 cm. Adjust the length when needed.</li>
	<li>Plug the silicone tube sections from step 2.2 into the ports of the radiator, pump tank, and copper cooling block, as shown in Figure 3B. Ensure all the connections are watertight. The water-cooling assembly is now built.</li>
	<li>Prepare the water-cooling assembly, a 12 V power supply, three red and three black jumper wires, a breadboard, and 500 mL of purified water.</li>
	<li>Ensure the workbench is clear of liquid for electrical safety.</li>
	<li>Connect the pump tanks and the radiator&#39;s wires to the 12 V power supply through the breadboard (Figure 3C). The breadboard is used for convenience. 
	NOTE: For a more permanent and safe connection, researchers can replace the breadboard with soldering wires.</li>
	<li>Open the pump tank cap using a flathead screwdriver. Use a funnel to add water until the pump tank is about 80% full (Figure 3D). Do not cap the pump tank after this filling.</li>
	<li>Power up the water-cooling assembly by plugging in the 12 V power supply or switching it on (if a switch is present). After powering up, the water will flow inside of the assembly and the fans on the radiator should blow.</li>
	<li>Due to the water flow from the pump tank, the liquid level in the tank will drop.&#160;Add more water to the pump tank until it stabilizes at nearly 2/3 full (Figure 3E).</li>
	<li>Shake the radiator to get rid of air bubbles and then cap the cooling tank.</li>
	<li>Turn off the power supply before going to the next step.</li>
</ol>
3. Testing Peltier cold and hot surfaces
NOTE: The Peltier, a key component of the cooling stage, is a solid-state active heat pump that transfers heat from one side to the other21. One surface of the Peltier becomes hot, and the other surface becomes cold when providing electric power. By default, Peltier manufacturers mark the cold surface before selling, but it is still helpful to manually test it before assembling.
<ol>
	<li>Prepare the tunable power supply and the Peltier, as shown in Figure 4A.</li>
	<li>Ensure the tunable power supply is off to prevent possible electrical hazards.</li>
	<li>Connect the red wire of the Peltier to the positive output and the black wire to the negative output of the tunable power supply with alligator clips, that are provided with the power supply (Figure 4B).</li>
	<li>Turn on the tunable power supply and set it to around 2 V by modulating both the voltage and current knobs on the top row of the power supply. Immediately use a bare finger to feel the two surfaces of the Peltier. One surface becomes cold within a few seconds.</li>
	<li>After identifying which surface is cold, immediately turn off the power supply and disconnect the Peltier.</li>
	<li>Use a marker to indicate the cold surface for future assembly.</li>
</ol>
4. Constructing the assembly to cool the Peltier using water-cooling assembly
<ol>
	<li>As shown in Figure 4A, prepare the switched-off water-cooling assembly, the Peltier (cold surface marked), and the thermal paste (for improved thermal conduction).</li>
	<li>Clean all the surfaces of the copper cooling block with 70% ethanol (or other cleaner solution) in the water-cooling assembly.</li>
	<li>Apply around 0.4 g of thermal paste to one surface of the copper water-cooling block and ensure this surface orientation will prevent the tubes from crossing or bending when facing down. Use a glove to protect the skin and try to distribute the thermal paste thinly and evenly (Figure 4C).</li>
	<li>Similarly, clean the hot surface of the Peltier, then apply the thermal paste to the surface (Figure 4D).</li>
	<li>Connect the Peltier hot surface to the copper cooling block surface with thermal paste. Apply pressure to ensure it is secure. Follow the orientation of the wires on the Peltier and the tubes of the copper cooling block, as shown in Figure 4E. Clean the excess thermal paste.</li>
	<li>Keep both the 12 V power supply and the tunable power supply off. Connect the Peltier to the tunable power supply, as in&#160;section 3.</li>
	<li>Recheck both the electrical and water-cooling assembly connections, and then turn on the 12 V power supply and tunable power supply sequentially.</li>
	<li>Gradually turn the tunable power supply to 12 V. With the suggested Peltier, the current should be around 7.3 A.</li>
	<li>Wait for 2 min; the temperature of the Peltier cold surface should become colder than -35 &#176;C. Measure this temperature with an infrared thermometer (Figure 4F). Do not touch the cold surface to prevent injury to the hands.</li>
	<li>Check all the connections and components if the temperature cannot reach below -30 &#176;C. Air bubbles inside the water-cooling assembly is a possible reason for suboptimal cooling performance.</li>
	<li>To ensure safety in later steps, turn off the tunable power supply, wait 1 min, then turn off the 12 V power supply.</li>
</ol>
5. Constructing copper plate and sapphire window assembly
<ol>
	<li>Prepare the copper plate, the 80 mm diameter sapphire window, thermal paste, a 4 inch wide tape, and a sharp blade for cutting (Figure 5A).</li>
	<li>Carefully clean the copper plate and the sapphire window with 70% ethanol and use fine-grit sandpaper to smooth rough surfaces.</li>
	<li>Apply the thermal paste on three inner surfaces, as shown in Figure 5B. Ensure the thermal paste covers all three surface areas but is not too thick, around 0.5 mm.</li>
	<li>Lay the copper plate on the benchtop protected with printer paper. The paper makes the later cleanup easier.</li>
	<li>Insert the sapphire window into the copper plate hole (Figure 5C). Ensure the sapphire does not rotate during insertion to prevent the thermal paste from moving to other areas. Remove excess thermal paste.</li>
	<li>Adhere the 4 inch wide tape to the top surface of the copper plate-sapphire window assembly (the surface that has the square depression area, as shown in Figure 5D). Avoid air bubbles between the tape and copper surfaces during pasting by guiding the adhesion slowly from one side to the other.</li>
	<li>Cut out the specified blue dashed areas of the tape using a sharp blade, following Figure 5E. Cutting exposes the two thread holes, the square depression, and 70 mm diameter area of the sapphire window.</li>
	<li>Tape the bottom surface of the copper plate-sapphire window assembly and then repeat the cutting procedure (sapphire area only) on this surface, as shown in Figure 5F. 
	&#8203;NOTE: Now, the sapphire window is fixed to the copper plate, and the copper surfaces are protected from rust.</li>
</ol>
6. Cooling stage final assembling
<ol>
	<li>Ensure all essential sub-assembly and components are ready.</li>
	<li>Apply about 0.4 g of thermal paste to the square depression of the copper plate (Figure 6A).</li>
	<li>Apply about 0.4 g of thermal paste to the cold surface of the Peltier. Note that the Peltier is already attached to the copper cooling block (Figure 6B).</li>
	<li>Connect the Peltier cold surface to the copper plate depression with downward pressure. Clean all excess thermal paste (Figure 6C).</li>
	<li>Mount the 3D-printed bracket on the top of the copper cooling block, and then use a hex key to tighten two 8-32, 0.5 inch long screws to fix the bracket to the copper plate (Figure 6D). Use low torque tightening so that the printed bracket does not break or deform to ensure proper thermal conduction from the Peltier to the copper.</li>
	<li>Place the copper plate into the 3D-printed isolation base for thermal isolation from the benchtop or microscope base during operation (Figure 6D).</li>
	<li>The cooling stage is assembled and ready to use (Figure 6E).</li>
	<li>For microscopy, place the completed cooling stage on an upright microscope platform (Figure 7A).</li>
	<li>Assembly of the cooling stage is completed. Further details are available from the Chung Laboratory&#39;s companion publication, which fully characterize the detailed strategies and animal movement20.</li>
</ol>
&#8203;NOTE: In the following sections, slow, fast, and abrupt cooling protocols are discussed. N2 hermaphrodites at L4 or young adult age were used to produce the following data. The slow cooling strategy is useful for immobilizing 20 &#176;C cultivated N2 animals at 6 &#176;C; 15 &#176;C-cultivated N2 animals are most strongly immobilized at 1 &#176;C20. A brief comparison between these three cooling protocols is shown in Table 1.
7. Slow cooling immobilization protocol
<ol>
	<li>Move the cultivation plate with a lid on to a 4 &#176;C refrigerator.</li>
	<li>After moving the cultivation plate to the refrigerator, turn on the cooling stage&#39;s 12 V power supply and set the tunable power supply voltage to 5.5 V.</li>
	<li>After the lidded cultivation plate has stayed in the 4 &#176;C refrigerator for 1 h, transfer the plate immediately to the cooling stage and remove the lid (Figure 7A). Such cultivation plates are usually around 6 &#176;C. The pre-cooled stage is stable and cold enough to maintain the agar surface at 6 &#176;C.</li>
	<li>If the agar surface temperature changes, as measured or by noting animal movement, slightly adjust the voltage until it stabilizes at 6 &#176;C.</li>
	<li>Animals are properly immobilized at the time of transfer.</li>
</ol>
8. Fast cooling immobilization protocol
NOTE: The fast cooling strategy is the most basic immobilization method (see Movie 1); however, agar plates idly occupy the stage for an extended time while reaching the Tset. Also, when a strong immobilization is needed and the Tset is 6 &#176;C, the idle time is extended to around 1 h20.
<ol>
	<li>Turn on the cooling stage&#39;s 12 V power supply and set the tunable power supply voltage to around 12 V. Wait for 10 min.</li>
	<li>Bring a cultivation plate from its incubator directly to the cooling stage and remove the lid.</li>
	<li>Once the agar surface temperature decreases to (Tset + &#916;T) &#176;C, adjust the tunable power supply to Vset and wait until the agar reaches Tset.&#160;The&#160;Vset is the appropriate voltage to stabilize the agar at Tset. The &#916;T is a variable that prevents overcooling. See Table 2 for the combination of Tset,&#160;&#916;T, and Vset. 
	NOTE: The data presented in Table 2 pertains specifically to the Chung Laboratory, and therefore it should be noted that experimental parameters may vary based on the unique environmental and usage conditions of each individual experiment.</li>
	<li>Animals are immobilized when the agar reaches Tset. Immobilization improves with time until ~50 min after the initiation of cooling.</li>
</ol>
9. Abrupt cooling immobilization protocol
NOTE: The abrupt cooling strategy consumes the most user time but immobilizes animals the most rapidly from their cultivation temperature.
<ol>
	<li>Turn on the cooling stage&#39;s 12 V power supply and turn the tunable power supply voltage to around 12 V. Keep for 10 min.</li>
	<li>Bring an unoccupied agar plate to the cooling stage. Use step 8.3 in the fast cooling immobilization protocol to stabilize the agar surface temperature at Tset.</li>
	<li>Move the animals from their original cultivation plate to the cooled plate sitting on the cooling stage.</li>
	<li>Based on the small animal size, the animals are expected to cool to Tset in seconds and be immobilized. Immobilization improves with time until ~50 min after the initiation of cooling.</li>
</ol>
10. Revival of animals after cooling immobilization
<ol>
	<li>Move the cooled culture plate back to the original incubator or to room temperature.</li>
	<li>Wait for 20 min to 1 h until all the worms on the plate revive to their normal crawling and feeding behavior.</li>
</ol>

Constructing and Operating a Cooling Stage to Immobilize C. elegans En Masse

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The authors declare no competing financial interests or other conflicts of interests.

The cooling stage manufacturing, assembling, and usage is shown in this manuscript. Most of the components are off-the-shelf items that can be purchased online. Some components, like the copper plate and the sapphire window, need a custom order and may take up to 1 month to fabricate. Other components that can be 3D-printed are easily fabricated in most research institutions (Supplementary Table 1). The assembling process needs only a few tools and can be quickly done by a non-expert in a few hours. Thus, most biological laboratories should be able to easily implement this device.
The cooling stage and the cooling immobilization approach possess several significant improvements over existing immobilization methods, carefully detailed in the original publication20. In brief, the cooling stage enables the strong immobilization of large populations of C. elegans of all ages, including embryos and dauers, on their typical culture plates under standard microscopy workflows. It eliminates the need for complex hardware setups, like microfluidics, while providing a stronger immobilization effect. Additionally, it minimizes the possible toxic chemical exposure to animals and researchers since no chemicals are used, while providing a similar immobilization effect. These technical capabilities enable the broad application of this device and approach to many experiments that require high-resolution in vivo microscopy on large numbers of animals.
There are some critical steps during the building of the device, including all thermal paste application and the wide tape to fix the sapphire window to the cooper plate. The thermal paste ensures strong thermal conductivity by replacing gaps with a low-thermal resistance material. To achieve the desired cooling performance, the paste needs to be properly introduced between all abutting/contacting surfaces, including the Peltier cold surface to the copper plate, the Peltier hot surface to the copper cooling block, and the copper plate to the sapphire window. The wide tape applied to the stage isolates the copper plate to prevent heating from air and condensation, which leads to rust. It also strengthens the connection between the sapphire window and the copper plate. Thus, both applying thermal paste and the wide tape require extra care.
In an actual cooling immobilization experiment, the parameters provided in this manuscript, such as voltages and times, depend on the specific properties of the cultivation plates and stage, such as the amount of agar in the plates, the stage&#39;s efficiency, and the ambient temperature and humidity. In future modifications, a feedback controller could be installed, like a proportional-integral-derivative (PID), to actively adjust the voltage input to the cooling stage to achieve desired temperature and stabilize it.
There are several limitations of this cooling stage immobilization, carefully detailed in the original publication20. In brief, animals raised at different temperatures are immobilized to different degrees, which may need extra fine-tuning. Also, this current cooling stage is not designed for an inverted microscope. Further, imaging or screening on a cultivation plate directly may introduce contamination to the plate.
We are designing new versions of the cooling stage suitable for different imaging platforms, including compound upright microscopes and inverted microscopes. These new designs will allow direct animal cooling immobilization on culture plates during imaging on these platforms. The imaging on these cooling stages will use long working distance air immersion objectives, similar to the upright configuration. Nowadays, air immersion objectives can have a numerical aperture of up to 0.9, which provide around a 300 nm resolution for green fluorescence protein imaging. Thus, the combination of a new cooling stage with a microscope could permit submicron resolution fluorescence imaging routinely.
We also provide some helpful tips for using the cooling stage according to our experience. For instance, individuals should check whether there are any air bubbles inside the water-cooling assembly. Air bubbles degrade the cooling to the Peltier hot surface and thus degrade the cooling effectiveness of the cooling stage. If air bubbles are present, the 12 V power supply should be turned on to make the water flow and all the components of the water flow should be shaken. Air bubbles can be flushed out from trapped areas and vented by the pump tank. Researchers should ensure that the water flow tubing is not bent or crossed when assembling the water-cooling assembly. Tube bending or crossing may prevent the adequate flow of water and reduce cooling efficacy. Tube connections should be properly fit and tight. If necessary, a soft tube with a different diameter can be used instead to ensure tightness. Paste should not be applied, even if the connection is not tight enough, as paste may introduce clogs during future usage. The room humidity affects the cooling performance and introduces condensation and ice on the cooling stage. Before placing a cultivation plate on the cooling stage, it is recommended to use a paper tissue to remove condensation or use a heatsink to quickly remove ice that has formed on the sapphire window. The pump tank and radiator fans can cause small vibrations in the microscope if they work on the same table. Microscope vibration blurs the image acquired and thus should be avoided. A cushion can be used to mechanically insulate the tank and radiator, or they can be placed on a separate nearby table. The cooling stage can become a heating stage by reversing the electrical connection to the Peltier.

High-resolution optical microscopy provides an indispensable tool for studying in vivo biological structures at the subcellular level. Many biological studies require submicron resolution imaging to resolve subtle anatomical details, including neuron morphology1,2, membrane structure3,4, and protein localization5,6. A high-resolution image requires an exposure time of several milliseconds to seconds, depending on the imaging modality and probe7,8. To achieve optimal results, it is essential to carefully plan and conduct microscopy-based experiments. Crucial to this effort is an efficient animal preparation method that facilitates high-resolution imaging.
The nematode C. elegans is a widely utilized model organism for studying many biological processes9. This small animal is typically cultivated on nematode growth medium (NGM) agar plates, and they reproduce rapidly by self-fertilization, making them well-suited for large-scale studies. Their transparency and a wide array of labeling techniques allow the straightforward visualization of their internal anatomy10,11. The fine structures in C. elegans are ideal for studying biological processes at the subcellular level, such as neuron regeneration12, neuron degeneration13, and cell division14. Such studies necessitate imaging at submicron resolution and animal immobilization strong enough to prevent image blur. Strong immobilization is especially crucial for techniques involving multiple images in space or time, such as 3D image stacks (i.e., z-stacks) and time-lapse imaging. Any animal movement between the exposures can obscure the result. For C. elegans, strong immobilization typically involves manual manipulation of individual animals and mounting them on slides with an anesthetic15,16. These time- and labor-intensive procedures make large-scale experiments very difficult. An immobilization strategy where animals are directly and reversibly immobilized on their original cultivation plates could enable high-throughput high-resolution imaging.
Cooling immobilization of C. elegans has been shown in a few studies but is not widely utilized. It is usually combined with a microfluidic device to further restrain animals17,18,19. However, microfluidic devices are complex, require significant operational training, and cannot be easily integrated with typical solid cultivation workflows of C. elegans experiments. Thus, microfluidics are not widely utilized for C. elegans immobilization. Presented here, in conjunction with the Chung Laboratory&#39;s recent publication20, is the introduction of a new cooling immobilization approach using a thermoelectric cooling stage (Figure 1) to address these shortcomings. With the cooling stage, a typical 60 mm polystyrene cultivation plate can be cooled down to any target temperature (Tset) between -8 &#176;C to room temperature. This cooling stage approach can readily and reversibly immobilize an entire animal population with minimal user effort, eliminating 98% of the animal processing time20.
Below, the procedures for building a cooling stage from scratch are described. Except for the machining of parts and 3D printing, the whole procedure is expected to take 4 h without the requirement of special tools or expertise. Then, three different cooling strategies with varying cooling rates and user efforts to immobilize C. elegans on a typical upright microscope are further described. The preferred strategy may depend on the user application. The protocols for those three cooling immobilization strategies are described in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-V power supply</td>
			<td>ANYTITI</td>
			<td>ledpower00</td>
			<td>output DC 12V +/-0.5V, 5A 
			power 60W</td>
		</tr>
		<tr>
			<td>8-32 screw</td>
			<td>arbitrary</td>
			<td></td>
			<td>for bracket fixation</td>
		</tr>
		<tr>
			<td>bracket</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>3D printed using 1.75mm PLA filament. See supplementary for 3D model.</td>
		</tr>
		<tr>
			<td>breadboard</td>
			<td>DEYUE</td>
			<td>7545924028</td>
			<td>400 pin solderless board kit for DIY electric connection</td>
		</tr>
		<tr>
			<td>copper cooling block</td>
			<td>Kalolary</td>
			<td>Kalolary-Heatsink001</td>
			<td>40*40mm 
			internal fin thickness 0.5mm</td>
		</tr>
		<tr>
			<td>copper plate</td>
			<td>arbitrary</td>
			<td>N/A</td>
			<td>Machined from a 170x120x3 mm 99.9% pure copper sheet. &#160;See supplementary for 2D drawing for manufacturing.</td>
		</tr>
		<tr>
			<td>digital thermocouple thermometer</td>
			<td>Proster</td>
			<td>4333090752</td>
			<td>dual channel thermometer with two K-type thermocouple probes 
			measuring range -50-300&#176;C 
			accuracy &#177;1.5% 
			resolution 0.1&#176;C /&#176;F &#60; 1000&#176;</td>
		</tr>
		<tr>
			<td>isolation base</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>3D printed using 1.75mm PLA filament. See supplementary for 3D model.</td>
		</tr>
		<tr>
			<td>jumper wires</td>
			<td>arbitrary</td>
			<td></td>
			<td>for electronic connection</td>
		</tr>
		<tr>
			<td>multistage peltier</td>
			<td>DigiKey</td>
			<td>TEC1-12706</td>
			<td>thermoelectric cooling device 
			size 40*40*7.05 mm 
			Umax 16.1 V&#160; 
			Imax 8.5 A 
			&#916;Tmax @ Th 85&#176;C @ 27&#176;C 
			Qmax @ Th 51.6W @ 27&#176;C 
			resistance 1.65 &#937;</td>
		</tr>
		<tr>
			<td>Nalgene 50 Platinum-Cured Silicone Tubing</td>
			<td>ThermoScientific</td>
			<td>14-176-332E</td>
			<td>ultrasoft tube 
			durometer hardness Shore A, 50 
			inner diameter 1/4 in 
			outer diameter 9.5 mm</td>
		</tr>
		<tr>
			<td>packaging tape</td>
			<td>arbitrary</td>
			<td></td>
			<td>4 inch wide to cover the copper plate</td>
		</tr>
		<tr>
			<td>pump tank</td>
			<td>Yosoo</td>
			<td>SC-300T</td>
			<td>input power DC 12V 
			flow rate 300L/h max</td>
		</tr>
		<tr>
			<td>radiator</td>
			<td>DIYhzWater</td>
			<td>10463</td>
			<td>12 pipe aluminum heat exchanger cooling water drain row with two 120mm fans</td>
		</tr>
		<tr>
			<td>sapphire window</td>
			<td>Altos Photonics, Inc.</td>
			<td>N/A</td>
			<td>Contact Altos for custom order 
			size &#216; 80mm, 3mm thick 
			surface quality 60-40s/d 
			uncoated</td>
		</tr>
		<tr>
			<td>thermal paste</td>
			<td>Corsair</td>
			<td>XTM50</td>
			<td>reduce thermal impedance between surfaces 
			thermal conductivity 5.0W/mK</td>
		</tr>
		<tr>
			<td>tunable power supply</td>
			<td>Kungber</td>
			<td>DY-SPS3010B</td>
			<td>voltage range 0 &#8211; 30V 
			current range 0 &#8211; 10A 
			linear Power Supply with 4-Digits 
			coarse and fine adjustments with alligator leads</td>
		</tr>
	</tbody>
</table>

assembly and operation of a cooling stage to immobilize c. elegans on their culture plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), but require strong animal immobilization to prevent motion blur in the images. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. Immobilization of C. elegans is greatly simplified by using a cooling approach that can easily immobilize entire populations directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures with a uniform distribution on the cultivation plate. In this article, the whole process of building the cooling stage is documented. The aim is that a typical researcher can build an operational cooling stage in their laboratory following this protocol without difficulty. Utilization of the cooling stage following three protocols is shown, and each protocol has advantages for different experiments. Also shown is an example cooling profile of the stage as it approaches its final temperature and some helpful tips in using cooling immobilization.

Cooling temperature measurement 
For the initial cooling immobilization experiments, it is important to track the agar surface temperature to ensure the animals can be properly immobilized. Future experiments that are replicated from the initial one can utilize the same parameters, usually without frequent temperature tracking. For temperature measurement, the thermocouple tip of the thermometer is sterilized using 70% ethanol solution, waiting until the ethanol fully evaporates before using. Then, the thermocouple tip is inserted 1 mm into the NGM agar to ensure an accurate temperature reading. The thermometer tip is held using a clamp holder or other holders (Figure 7B).
Temperature measurement with an infrared camera 
The cooling stage is designed to ensure the temperature distribution in the plate&#39;s central 40 mm diameter area is uniform. A forward-looking infrared (FLIR) camera is used to image the temperature distribution on the agar surface. The maximum temperature difference is around 1 &#176;C when the Tset is 1, 3, or 6 &#176;C (Figure 8A).
Assessing the cooling rate with the fast cooling strategy 
The fast cooling strategy is used to characterize the cooling rate of a stage at 12 V. A 20 &#176;C plate is placed on the cooling stage and a thermocouple thermometer is used to track the surface temperature. The stage cools down the 20 &#176;C plates to 6 &#176;C in 6 min, to 1 &#176;C in 10 min, and eventually stabilizes below -7 &#176;C in around 40 min (Figure 8B).
Using the cooling stage on an upright microscope platform 
An upright microscope typically comprises an objective for imaging, a stage for sample holding, and illumination. This cooling stage is designed for use on a typical upright microscope stage with easy insertion and removal (Figure 8C). When cooling immobilization is needed for imaging or screening, the cooling stage is simply placed on the microscope stage to finish the installment and vice versa.
Immobilization of worms on the cooling plate is shown in Movie 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65267/65267fig01.jpg" /> 
Figure 1: 3D model of the cooling stage apparatus. Electronic connections are not shown for clarity. A tank pumps water through the cooling block to remove heat transferred by the Peltier embedded in the stage. A typical 60 mm polystyrene cultivation plate can sit on the transparent sapphire window and be cooled by stage. Model generated in Solidworks. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig01large.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/65267/65267fig02.jpg" /> 
Figure 2: 3D models of components to be manufactured. (A) Copper plate. (B) 3D-printed holding bracket. (C) 3D-printed isolation plate. Models generated in Solidworks. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig02large.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/65267/65267fig03.jpg" /> 
Figure 3: Water-cooling assembly. (A) Individual components. Tubes cut to specified lengths. (B) Water-cooling components connected. (C) Wires connecting the pump tank and the radiator to the 12 V power supply. In general, red wires connect to positive end, and black wires to the negative end. (D) Purified water poured into the pump. (E) The tank filled to more than two-thirds for optimal pump efficiency. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig03large.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/65267/65267fig04.jpg" /> 
Figure 4: Connecting the Peltier and water-cooling assembly. (A) Components to operate the Peltier. (B) Utilizing the tunable power supply to determine the hot and cold sides of the Peltier. For safety, no more than 2 V is used. (C) Even application of thermal paste to the surface of copper block. (D) Even application of thermal paste to the Peltier hot surface. (E) Hot side of the Peltier pressed onto the copper block with thermal paste. (F) Infrared thermometer used to measure the Peltier cold surface temperature. Ideally, the cold temperature can reach near -35 &#176;C. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig04large.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/65267/65267fig05.jpg" /> 
Figure 5: Assembling the copper plate and sapphire window. (A) Components required. (B) Thermal paste applied to three inner surfaces of the copper plate where the sapphire window will contact. Two downward-looking views of the copper plate showing the location of the three surfaces. (C) Sapphire window in the copper plate hole. (D) Tape applied to the top surface of the assembly. (E) Top-side: Blue dashed lines indicate the locations to cut and remove tape: square depression, two holes, and a 70 mm diameter sapphire area. (F) Bottom-side: Tape is cut and removed as shown. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig05large.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/65267/65267fig06.jpg" /> 
Figure 6: Cooling stage final assembly. (A) Thermal paste applied to the depression of the copper plate. (B) Thermal paste applied to the cold side of the Peltier. (C) Cold surface of the Peltier connected to the depression. (D) Copper cooling block fixed to the copper plate using screws. Cooling stage in the isolation base. (E) Completed cooling stage. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig06large.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/65267/65267fig07.jpg" /> 
Figure 7: Cooling stage on the microscope and thermocouple measurement. (A) Cooling stage placed on the microscope base for imaging. The sapphire window is transparent, allowing transillumination. (B) Thermocouple thermometer used to measure the NGM agar surface temperature. The tip inserted around 1 mm into the NGM agar. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig07large.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/65267/65267fig08.jpg" /> 
Figure 8: Cooling stage characterization and usage. (A) Thermal images showing the agar surface cooled to 1, 3, and 6 &#176;C. Even temperature distribution within the central 40 mm area (white dashed circle). (B) Temperature of the NGM agar surface over time on the cooling stage at 12 V. The NGM agar surface can be cooled below -7 &#176;C. Temperature measured by the method in Figure 7B. (C) Cooling stage in use on a typical upright microscope. The cooling stage can be easily installed or removed. <a href="https://www.jove.com/files/ftp_upload/65267/65267fig08large.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></td>
			<td>slow cooling</td>
			<td>fast cooling</td>
			<td>abrupt cooling</td>
		</tr>
		<tr>
			<td>stage occupation</td>
			<td>minimum</td>
			<td>long</td>
			<td>medium</td>
		</tr>
		<tr>
			<td>time until animals are immobilized</td>
			<td>long</td>
			<td>medium</td>
			<td>very short</td>
		</tr>
		<tr>
			<td>immobilization strength</td>
			<td>strong</td>
			<td>medium</td>
			<td>medium</td>
		</tr>
		<tr>
			<td>user effort</td>
			<td>minimum</td>
			<td>slightly more than minimum</td>
			<td>maximum</td>
		</tr>
	</tbody>
</table>
Table 1: Cooling strategies comparison.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tset (&#176;C)</td>
			<td>&#916;T (&#176;C)</td>
			<td>Vset (V)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>8</td>
		</tr>
		<tr>
			<td>2</td>
			<td>3</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>3</td>
			<td>4.5</td>
			<td>7</td>
		</tr>
		<tr>
			<td>4</td>
			<td>5.5</td>
			<td>6.5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>6</td>
			<td>5.9</td>
		</tr>
		<tr>
			<td>6</td>
			<td>6</td>
			<td>5.5</td>
		</tr>
	</tbody>
</table>
Table 2: Parameters to achieve the desired temperature in the fast cooling strategy.
Supplementary File 1: Copper plate in metric. A2D drawing for machining the copper plate. <a href="https://www.jove.com/files/ftp_upload/65267/Copper plate in Metric.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Holding bracket. A 3D drawing of a holding bracket that can be opened or modified by Solidworks and exported to 3D printing software. <a href="https://www.jove.com/files/ftp_upload/65267/holding bracket.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Isolation plate. A 3D drawing of an isolation plate that can be opened or modified by Solidworks and exported to 3D printing software. <a href="https://www.jove.com/files/ftp_upload/65267/isolation plate.zip" target="_blank">Please click here to download this File.</a>
Movie 1: Cooling video. Immobilization worms on the NGM agar plate at 2 &#176;C. The plate was cooled down from room temperature to 2 &#176;C, and stayed at 2 &#176;C for several minutes. Then, the cooling stage was turned off and plates began to warm up to room temperature naturally. The video is speeded up by 10x to fit a 1 h video in 6 min. <a href="https://www.jove.com/files/ftp_upload/65267/Cooling video.mp4" target="_blank">Please click here to download this Movie.</a>
Supplementary Table 1: Price estimation <a href="https://www.jove.com/files/ftp_upload/65267/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates at JoVE.com

Author Spotlight: Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates  

This paper describes protocols for constructing and operating a cooling stage to immobilize C. elegans on their original cultivation plates en masse.

Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), ...

assembly-operation-cooling-stage-to-immobilize-c-elegans-on-their

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2.4K Views.  Northeastern University. This paper describes protocols for constructing and operating a cooling stage to immobilize C. elegans on their original cultivation plates en masse. 

Video: Author Spotlight: Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates  

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 ...

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

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 ...

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

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 ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...