This work was supported by the National Institutes of HealthGrants DK85035 and DK074797 (I.H).

1. Worm Strains
<ol>
	<li>Obtain C. elegans wildtype Bristol N2 strains from the Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc) and maintain them on NGM plates seeded with E. coli strain OP507. Note: Transgenic worm strains IQ6011 (Phrg-1::GFP) utilized were generated as previously described11. IQ6011 can be requested from the corresponding author.</li>
</ol>
2. Preparation of Modified C. elegans Habitation and Reproduction Medium (mCeHR)
Prepare mCeHR liquid media as described below12. This axenic liquid media allows the worms to grow without any additional food sources. Carry out all manipulations of axenic liquid media and axenic worms using strictly sterile conditions such as a laminar flow hood.
<ol>
	<li>Obtain ultra pasteurized fat-free milk from a grocery store. Use the refrigerated product and not the room temperature product. Prior to use in mCeHR medium, streak the milk on Brain Heart Infusion (BHI) agar plates and incubate for 3 days at 30 &#176;C and 37 &#176;C to check for sterility. Transfer the milk to 50 ml sterile tubes and store at -80 &#176;C</li>
	<li>Prepare each of the following components and combine in the order and amounts given to prepare 1 L&#160;of mCeHR (Table 1A): 10 ml of 2 mM choline diacid citrate, 10 ml of vitamin and growth factor mix (see recipe in Table 1B), 10 ml of 2.4 mM myo-inositol, 10 ml of 2 mM hemin chloride, 250 ml of deionized water. Apply suction and filter through a 0.22 &#956;m filter unit.</li>
	<li>Add 20 ml of nucleic acid mix (recipe in Table 1C), 100 ml of mineral mix (recipe in Table 1D), 20 ml of 170 mg/ml lactalbumin hydrolysate, 20 ml of essential amino acids, 10 ml of non-essential amino acids, 20 ml of 450 mM KH2PO4, 50 ml of 1.45 M D-glucose, 10 ml of 1 M HEPES, sodium&#160;salt, 250 ml deionized water.</li>
	<li>Apply suction to filter sterilize the components. Remove the filter and add 1 ml of 5 mg/ml cholesterol. Ensure that the pH of the media is approximately 6-6.5. Note: This solution can be stored at -80 &#176;C for up to one year.</li>
	<li>Add 20% (v/v) ultra pasteurized organic skim milk to the mCeHR medium before use. Use scrupulously sterile technique (for example in a laminar flow hood) when opening and aliquoting the milk. Store the media at 4 &#176;C.</li>
</ol>
3. Prepare C. elegans for Culture in Axenic mCeHR Liquid Medium
<ol>
	<li>Grow worms on ten 60&#160;mm NGM plates until there are many gravid worms and a minimal amount of OP50 E. coli on the plates.</li>
	<li>Remove the worms from each plate by rinsing with 5 ml of M9 buffer (recipe in Table 1F), and combine in a 50 ml conical tube.</li>
	<li>Allow the worms to settle by leaving the tube to stand for 5 to 10 min&#160;then carefully remove the supernatant containing the E. coli.</li>
	<li>Repeat steps 3.2 and 3.3 2x to remove as much of the residual bacteria as possible before continuing the procedure.</li>
	<li>Resuspend the worms in 0.1 N NaCl in a volume that is a multiple of three, for example 1.5 ml, 3 ml or 6 ml in which individual worms can be seen in the tube.</li>
	<li>Bleach the worms by adding 5 N NaOH and 5% sodium hypochlorite (bleach) solution in a 1:2:6 ratio to the worm suspension. For example, 1 ml 5 N NaOH: 2 ml 5% sodium hypochlorite: 6 ml of the worm suspension. Mix the NaOH and bleach before adding to the worm suspension.</li>
	<li>Vortex the solution vigorously until the gravid worms are dissolved and only eggs are visible within the suspension (about 5 to 10 min). Monitor the process using a phase contrast microscope with a 10X objective.</li>
	<li>After the worms have completely dissolved, immediately pellet the eggs at 800 x g for 45 sec at 4 &#176;C. Remove the supernatant and rinse the pellet twice with 10 ml of sterile water by centrifuging the pellet at 800 x g for 45 sec at 4 &#176;C.</li>
	<li>After bleaching, transfer the egg pellet to a 25 cm2 tissue culture flask containing 10 ml of mCeHR media supplemented with 100 &#956;g/ml tetracycline. Carry out this procedure using sterile techniques. If desired, add additional antibiotics, including 250 &#956;g/ml nalidixic acid, to prevent bacterial growth in the axenic liquid cultures.</li>
	<li>Incubate the liquid cultures at 20 &#176;C on a shaking incubator at approximately 70 rpm.</li>
	<li>Check the worms daily to note the stage of development and the rate of growth. 
	Note: Worms will initially grow slowly and require 7 to 10 days to become gravid. However, as the worms acclimatize to the liquid medium the generation time will decrease to approximately 4 days for wildtype (N2) worms.</li>
	<li>After the worms have developed to the gravid stage in liquid media, transfer the worm culture to a conical tube and pellet the worms at 800 x g for 5 min&#160;at 4 &#176;C using a swinging bucket rotor.</li>
	<li>Remove the supernatant and gently resuspend the worm pellet in an equal volume of 0.1 M NaCl. For example, use 10 ml of 0.1 M NaCl for worms cultured in 10 ml of mCeHR. Allow the worms to settle for 5 min&#160;on ice.</li>
	<li>Carry out the bleaching procedure as described above in steps 3.5-3.8, and allow the worms to grow for a second generation in the axenic liquid media. If necessary, add the antibiotic again to inhibit bacterial growth.</li>
	<li>Again allow the worms to become gravid then pellet and resuspend the worms in 0.1 M NaCl as described in steps 3.12 and 3.13, then bleach again as described in steps 3.5-3.8, to produce a synchronized population. Grow the L1 larval worms in liquid media without antibiotics from now on.</li>
</ol>
4. Synchronizing Worms from Liquid Culture
<ol>
	<li>Pellet and resuspend the worms in 0.1 M NaCl as described above in steps 3.12 and 3.13. Bleach as described above in steps 3.5-3.8.</li>
	<li>Resuspend the egg pellet in 10 ml of M9 buffer and allow the larvae to hatch O/N at 20 &#176;C on a rotating platform shaker. Note: The eggs will hatch into L1 larvae, but will not develop further, synchronizing the worms at the L1 stage.</li>
	<li>To maintain and subculture worms, pellet L1 larvae at 800 x g for 5 min, then resuspend the larvae in 10 ml of mCeHR and transfer to a 25 cm2 flask. Allow the worms to grow at 20 &#176;C on a rotating platform. Maintain a maximum density of 3,000 worms/ml/cm2 to ensure adequate nutrition for the worms. 
	Note: At this stage, antibiotics are not required to maintain axenic conditions when the procedures are carried out using sterile techniques in a laminar flow hood. Worms should be checked daily to monitor growth.</li>
</ol>
5. Freezing and Thawing Worms for Axenic Medium Cultures
<ol>
	<li>Freeze axenic asynchronous worm cultures or synchronized L1 larvae in liquid nitrogen. Pellet worms at 800 x g for 5 min then resuspend in S buffer (6.45 mM KHPO4, 43.55 mM KHPO4, 146.25 mM NaCl) using 0.5 ml for each vial. Add an equal volume of S buffer with 30% v/v glycerol to each vial and transfer the worms to -80 &#176;C before long term storage in liquid N2. Freeze approximately 1 x 106 worms per ml in each vial.</li>
	<li>Thaw worms by incubating the vials at 37 &#176;C for approximately 2 min until almost all of the ice is melted. In a laminar flow hood, transfer the worms to a sterile flask containing mCeHR and place them at 20 &#176;C.</li>
</ol>
6. Determine the Effect of Hemin Concentration on Growth and Reproduction in mCeHR
<ol>
	<li>Use axenic mCeHR media to assay nutrient dependent growth of C. elegans. To determine the effect of hemin concentration on worm growth and development, use synchronized axenic N2 worms or other strains of interest. Synchronize L1 larvae as described above in section 4.</li>
	<li>On the following day, pellet and count synchronized L1 larvae and dilute to 1 worm per &#956;l of axenic media. Make 10 mM hemin chloride in 0.3 M NH4OH and adjust to pH 8 using concentrated HCl.</li>
	<li>Add 800 &#956;l of mCeHR media to a 24-well plate. Add 100 worms to each well in 100 &#956;l of media. Add hemin chloride to each well in concentrations ranging from 0 &#956;M, 1.5 &#956;M to 1,000 &#956;M in triplicate. Ensure that all wells contain the same concentration of 0.3 M NH4OH. Gently resuspended the worms before pipetting to ensure that 100 L1 larvae are added to each well.</li>
	<li>Examine the worms daily and note the growth in each concentration in each well. Count the number of worms after 9 days of incubation and plot the number of worms/ml corresponding to each heme concentration (Figure 1).</li>
</ol>
7. Effect of Hemin Concentration on Heme Sensor Worms
<ol>
	<li>Incubate synchronized L1 heme sensor worms (Phrg-1::gfp) in mCeHR containing 4 &#956;M, 8 &#956;M, 10 &#956;M and 20 &#956;M hemin.</li>
	<li>Image the worms using fluorescence confocal microscopy, 48 hr&#160;post incubation (Figure 2).</li>
</ol>
8. Utilizing mCeHR to Generate Transgenic C. elegans&#160;Using Microparticle Bombardment
Note: The procedure for generating and carrying out microparticle bombardment using unc-119(ed3) worms grown in mCeHR is outlined in Figure 313.
<ol>
	<li>Worm Preparation
	<ol>
		<li>Transfer approximately 1 x 106 asynchronous unc-119(ed3) worms in 90 ml of mCeHR media in a 175 cm2 flask one week prior to bombardment. Allow the worms to grow at 20 &#176;C on a shaking platform at ~70 rpm (Figure 3-1). 
		Note: One week later, the worm density should be significantly greater with many adult gravid worms. Approximately 3 x 107 worms will be required for microparticle bombardment.</li>
		<li>Chill JM109 E. coli seeded 10 cm NGM plates on ice.</li>
		<li>Transfer the axenic worms to two 50 ml conical tubes and centrifuge the worms at 800 x g for 2 min. Aspirate the supernatant and resuspend the worms in each tube in 50 ml of M9 buffer (Figure 3-2).</li>
		<li>Allow the adult worms to pellet by gravity for 10 to 15 min&#160;on ice. 
		Note: The 2 ml pellet should now contain &#62;90% adult worms. This 2 ml pellet should have approximately 5 x 106 worms and is sufficient for one microparticle bombardment.</li>
		<li>Coat the entire surface of the chilled JM109 seeded NGM plate with the 2 ml pellet of worms. Allow the liquid to be completely absorbed then leave the plate for 30 min&#160;on ice before proceeding (Figure 3-3).</li>
	</ol>
	</li>
	<li>Preparation of gold particles
	<ol>
		<li>Weigh 30 mg of gold particles into a siliconized microcentrifuge tube. Add 1 ml 70% ethanol and vortex the tube for 5 min.</li>
		<li>Allow the tube to sit for 15 min, then centrifuge at 6,000 x g for 10 sec to pellet the gold particles. Remove the supernatant using a pipette tip by carefully touching the tip to the side of the tube opposite to the gold particles.</li>
		<li>Wash the gold particles with 1 ml of sterile water, vortex for 1 min&#160;and briefly centrifuge the tube at 6,000 x g for 10 sec. Repeat the wash step twice, then resuspend the gold particles in 0.5 ml of sterile 50% glycerol. 
		Note: The final concentration of the gold particles will be 60 mg/ml and it can be stored for 1 to 2 months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the DNA-gold particle mix
	<ol>
		<li>Combine 10 &#956;g of the desired plasmid DNA with 10 &#956;g of the unc-119 rescue plasmid in a siliconized 1.5 ml microcentrifuge tube. Add 50 &#956;l of DI water and 100 &#956;l of well resuspended gold particle solution then vortex for 1 min.</li>
		<li>Add 150 &#956;l of 2.5 M CaCl2 and vortex the solution again for 1 min. To this solution, add 60 &#956;l of 0.1 M spermidine and vortex for 3 to 5 min.</li>
		<li>Pulse centrifuge the solution at 6,000 x g for 10 sec, remove the supernatant and add 300 &#956;l of 70% ethanol. Vortex well for 1&#160;min, pulse centrifuge at 6,000 x g, remove the supernatant and resuspend in 170 &#956;l of 100% ethanol. Vigorously vortex the solution for 5 to 10 min&#160;then pulse centrifuge and remove the supernatant.</li>
	</ol>
	</li>
	<li>Bombardment (Figure 3-3) 
	Note: This protocol is carried out with the particle delivery system specified in the Table of Materials/Equipment. Follow the instructions for the particle delivery instrument available.
	<ol>
		<li>Place a sheet of tissue paper into a 15 cm plate. Using tweezers, dip seven macrocarriers individually into 100% ethanol and lay them on the tissue to dry.</li>
		<li>Thoroughly clean the biolistic chamber, macrocarrier holder, and target plate with 70% ethanol. Clean and autoclave the stopping screens before each bombardment procedure.</li>
		<li>Load the macrocarrriers into the holder using tweezers and press into the holder using the seating tool.</li>
		<li>Evenly spread 20 &#956;l of the DNA coated gold particles in the center of each macrocarrier and then allow the solution to dry completely.</li>
		<li>Clean the two parts of the pressure divider with ethanol and assemble with ethanol cleaned rupture disks. Screw the assembled pressure divider and rupture disks into the bombardment chamber.</li>
		<li>Assemble an autoclaved stopping screen with the macrocarrier holder and place the apparatus on to the metal shelf required and lock in place.</li>
		<li>Insert the assembled macrocarrier apparatus into the biolistic chamber in the allotted slot below the pressure divider and align with the pressure divider.</li>
		<li>Tape the open NGM plate with the worms onto the target plate shelf and close the door of the chamber.</li>
		<li>To bombard worms, adjust the pressure to that needed to break the rupture disks. Turn on the vacuum to 28 inches of Hg pressure, then press the fire button until the disks rupture.</li>
		<li>Release the vacuum and remove the NGM plate from the apparatus. Wash the worms from the plate and redistribute in twenty 10 cm NGM plates seeded with JM109 E. coli (Figure 3-4).</li>
		<li>Allow the worms to grow for 10 to 14 days at 25 &#176;C and starve the plates. Worms rescued from the unc-119 defect will have normal movement and can be identified at this time. Pick at least 10 &#8220;wildtype&#8221; worms from separate plates for further analysis as these represent independent transgenic lines (Figure 3-5).</li>
	</ol>
	</li>
</ol>

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The authors declare there are no competing financial interests or conflict of interests.

In this protocol we present a modified axenic liquid media mCeHR that allows for rapid C. elegans generation with production of a large number of worms. This media shows several advantages as the worms are grown without contaminating E. coli or bacterial byproducts and can be exploited in nutritional and toxicological studies. The use of E. coli or other bacteria in such studies has several drawbacks. For example, the growth of the bacteria can change under various conditions and the bacteria m...

The soil nematode, Caenorhabditis elegans, is a powerful model organism used in numerous studies from genetics to toxicology. As a result of its 1 mm size, rapid generation time of four days, ease of cultivation, and large progeny numbers, these nematodes have been utilized in a number of genetic and pharmacological screens1,2. Researchers take advantage of this worm to identify molecules and pathways conserved in vertebrate systems. These pathways include cell death signals, pathways of aging and metabolism, and the nervous system3-6. Additionally, the transparency of C. elegans allows for the generation of transgenic lines using fluorescent protein reporters, which can be directly visualized to analyze gene expression patterns and protein localization.
In many studies this nematode is cultured on a solid agar-based surface using nematode growth medium (NGM) plates or in liquid cultures seeded with Escherichia coli as a food source7,8. These bacterial food sources can confound biochemical and toxicology studies with interference from bacterial by-products affecting the interpretation of results. In order to avoid these compounding effects, C. elegans can be cultured in an axenic liquid media that is devoid of bacteria as a food source. Using this media, we successfully cultured millions of highly synchronized worms for many standard C. elegans protocols including microarray analysis of differentially regulated genes in C. elegans exposed to different heme concentrations, and production of transgenic worms using gene bombardment. This media is chemically defined and modified from an original recipe formulated by Dr. Eric Clegg9. Using this mCeHR media, we have successfully identified genes involved in heme homeostasis, referred to as heme responsive genes (hrgs)10, which would have not been possible in regular growth conditions which utilize NGM agar plates seeded with E. coli.
In this protocol we describe the procedure for introducing and maintaining C. elegans grown on E. coli to the axenic mCeHR and utilize this method to obtain a large number of worms for producing transgenic C. elegans lines using microparticle bombardment. We also present studies that show the utility of using axenic media for determining the nutritional requirement of C. elegans using heme as an example. These studies show that using mCeHR media allows for rapid growth of a large number of C. elegans for many downstream applications utilized by worm researchers.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">MgCl2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">M-2393</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">S-4641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium citrate.H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-1722</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C455-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">M87-100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnCl2</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">Z-0152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fe(NH4)2(SO4)2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-1018</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C70-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5 -monophosphate, sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-1752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5 -phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine 2 - and3&#160; -monophosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine 5 -phosphate, disodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">U-6375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymine&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T0376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N-Acetylglucosamine</td>
			<td style="width:108px;">Sigma</td>
			<td>A3286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-Alanine</td>
			<td style="width:108px;">Fisher</td>
			<td>S25648&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-Aminobenzoic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-9878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Biotin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">B-4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cyanocobalamine (B-12)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">V-2876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Folinate (Ca)&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-7878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-0761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacinamide</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-3376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantetheine</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-2125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantothenate (Ca)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pteroylglutamic Acid (Folic Acid)</td>
			<td style="width:108px;">ACRCS</td>
			<td style="width:121px;">21663-0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxal 5&#39;-phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-3657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxamine.2HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6280</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Riboflavin 5-PO4(Na)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">R-7774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thiamine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-6,8-Thioctic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1395</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-5379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Choline di-acid citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">myo-Inositol</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">I-5125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-Glucose</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactalbumin enzymatic hydrolysate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">L-9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion</td>
			<td style="width:108px;">BD</td>
			<td style="width:121px;">211065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Hemin chloride</td>
			<td style="width:108px;">Frontier Scientific</td>
			<td style="width:121px;">H651-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HEPES, Na salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">H-3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cholesterol</td>
			<td style="width:108px;">J.T. Baker</td>
			<td style="width:121px;">F676-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11140-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids Solution</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalidixic acid sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td>N4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline Hydrochloride</td>
			<td style="width:108px;">MP Biomedicals</td>
			<td>2194542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biolistic Delivery System</td>
			<td style="width:108px;">BioRad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Gold particles (Au Powder)&#160;</td>
			<td style="width:108px;">&#160;Ferro Electronic Material Systems</td>
			<td style="width:121px;">6420 2504, JZP01010KM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">or</td>
			<td style="width:108px;"></td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Particles 1.0 &#956;m</td>
			<td style="width:108px;">BioRad</td>
			<td>&#160;165-2263</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing C. elegans in axenic liquid medium aids in the determination of nutrients that are required by worms, without interference from secondary metabolites produced by E. coli. Wildtype N2 worms acclimatize to mCeHR media within three generations and show growth comparable to worms grown on NGM bacterial plates. Indeed, these worms become gravid within 4&#160;days as compared with 3.5 days for worms grown on OP50 bacteria.
One advantage of using mCeHR was seen in studies ...

Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

culturing-caenorhabditis-elegans-axenic-liquid-media-creation

Research

JoVE Journal

Biology

16.9K visualizações.  University of Maryland.  C. elegans é normalmente cultivadas em placas de agar sólido ou em meio líquido semeadas com E. coli. Para evitar subprodutos bacterianos de confundir os estudos toxicológicos e nutricionais, utilizamos um meio líquido axênico, CEHR, para crescer e sincronizar um grande número de vermes para uma gama de aplicações a jusante. 

Vídeo: Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

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 ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...