(Modified from the laboratory manual for axenic cultivation of Caenorhabditis elegans, by Nancy C. Lu, Ph.D., R.D., Department of nutrition and food science, San Jose State University, San Jose, CA 95192-0058)* some modifications to methods are present)

* Autoclave and keep in refrigerator or in sterile bottles on shelf

When needed remove HLE from freezer and thaw.

Use 1ml of HLE for every 9ml HS-YE.

 Small cultures

• In previously autoclaved bottles containing 3 ml of HS-YE, add 0.5ml HLE
• Store bottles either upright or on edge.

 Large cultures of (HS-YE-HLE)

500ml Wheaton bottles containing 40ml of HS-YE and 4 ml HLE have been successfully maintained for ~3 months. Bottles do not need to be shaken. Stand them upright on a shelf.

 Setup of a 500ml of culture

• using 40ml HLE and 500ml HySoy in a 3 liter flask.
• The stopper of the flask was penetrated by a pipette plugged with cotton and the HS-YE was autoclaved in the flask.
• HLE was added through the pipette after flask cooled .
• Pipette was then closed with sterile cotton and covered in parafilm.
• Set on shelf and swirl every few days.

• Purchase 1-2 pounds of devained calf or beef liver from grocery store.
• Wash and cut into 1-inch squares.
• Drain and place in refrigerator for autolysis for approximately 24 hours.
• Place liver in an equal volume of water and homogenize in a blender until the large clumps have been removed.
• Filter through Miracloth and keep on ice until all liver has been homogenized.
• Place 600 ml of the homogenate into a 1-liter beaker and place the beaker in a 60 centigrade water bath.
• Stir constantly while keeping an eye on the homogenate temperature. A thermometer should be in the homogenate to allow observation of the temperature.
• Once temp has reached 52-53 C, keep temperature in range for exactly 6 min.
• Remove beaker and place the beaker into an ice water bath to reduce the homogenate temp. (Stirring will help reduce temp quickly).
• Place homogenate into capped centrifuge tubes and Centrifuge at 37,000-39,000 g, using an SS-34 rotor at 18,000 rpm( do not use brake).
• Collect all supernatant from the homogenate and filter it through Miracloth.
• Pre-filter extract with 1.0 micron Gelman science 61664 type A/E glass fiber 90mm. With a Buchner vacuum filter apparatus.
• Sterilize finally using Nagalene 125-0045 sterilization filter unit (either Cellulose Nitrate, or Nylon sterilizing filter) 0.45 micron, 150ml capacity reservoir.
• To check the sterility of: the HLE or the cultures use the same inoculation amount of nematode stock used when stocks are transferred, but place it in a tube with 2 ml Nutrient Broth-Yeast Glucose extract (NYG), incubate at 30C for 3-4 days checking occasionally.

 Method for continual subculture of Axenic Caenorhabditis elegans nematodes:

Stock cultures are kept as follows:

• 6-8 previously autoclaved parafilm wrapped, screw capped vials containing 3ml of HS-YE are placed in a laminar flow hood and sprayed with a 1:100 solution of Lysol-IC.
• Tubes are allowed to dry while inoculation tube and HLE is prepared.
• Remove HLE from the freezer.
• Spray outside of HLE container with Lysol-IC.
• Allow HLE to thaw in laminar flow hood.
• While HLE thaws, locate the culture to subculture and check the nematodes for movement.
• Place culture in laminar flow hood.
• Spray outside of culture container with Lysol-IC.
• Wearing gloves sterilized with Lysol-IC, remove parafilm and spray all surfaces of vials again with Lysol-IC.
• Remove caps of vials and place them in sterile covered petri dishes.
• Using a sterile cotton plugged pipette, remove 3-4ml of HLE from its container.
• Place 2-2.5ml in two of the HY-YE tubes.
• Inoculate the other HY-YE tubes with HLE by splitting the two new HLE-HY-YE tubes between the remaining tubes.
• Using new sterile cotton plugged pipette, withdraw all of the HLE-HY-YE from one of the tubes and replace only 3ml of the fluid.
• Replace the remaining fluid into one of the two empty HLE-HY-YE tubes.
• Repeat steps 14 and 15 until all HLE-HY-YE tubes have approximately the same volume.
• Remove 1.00ml of from the selected HLE-HY-YE culture tube to be used for inoculation and place 4-20 drops into each new HLE-HY-YE tube.
• Replace caps onto tubes.
• Wrap tube caps in a clockwise fashion with parafilm .
• Label with ID# and date.

• Remove 0.25ml of HLE-HY-YE culture selected as inoculation culture.
• Place several drops on a slide or Petrie dish.
• Observe drops under a microscope.
• Choose an inoculation volume that will inoculate new cultures with at least 10 to 20 moving nematodes.

• After transfer of nematodes from HLE-HY-YE media to CbMM media, determine the mixed concentration of nematodes in 1,2,3&4 drop volumes of the media.
• Adjust the number of drops until the number of inoculation is around 20-30 nematodes.
• If the nematodes require more than 5 drops to reach a high enough concentration, then allow nematodes to settle and re-suspend them in a smaller volume of CbMM, or just remove some of the excess volume of CbMM after they have settled to the bottom of the tube, to allow for proper inoculation number.

Place `2ml into a quantity of small capped vials and autoclave at 110C-122C, 15 psi for 15 min..

Store in refrigerator or shelf.

Compound three salt solutions separately

To compound salt solution A

• In a 50 ml beaker.
• Add 10 ml water and stir until dissolved.

To compound Salt Solution B

• Place components in a 50 ml or 250ml beaker.
• Add 10 ml or 100ml water and stir until dissolved.

To Compound Salt Solution C

• Place Components in a 100ml or 400ml beaker.
• Add 20ml or 200ml water stir until dissolved.

• Combine in a 1 liter beaker Salt Solution B to Salt Solution C using no more than 1ml transfer water and stir.
• Add Salt Solution A (use no more than 1 ml transfer water) to Combined Salt Solutions BC and stir until dissolved.
• Store Frozen in 50ml capped vials.

To compound media WSC

• Weigh out above chemicals and place in 100 or 400 ml beaker.
• Add 20ml or 160ml of water and stir until dissolved.
• Transfer quantitatively to 25 or 200 ml volumetric flask, depending on amount to be made, and bring to volume.
• Separate into aliquots of about 8 ml each and store frozen.

To compound above media TSC:

• Weigh out above components and place in 100ml or 500ml beaker.
• Add 1.3ml or 13.00ml of 10% W/V aqueous solution of TEA.
• Add 19ml distilled water.
• Stir and heat to not more than 40C until dissolved.
• Bring to final volume in a 25ml or a 200ml volumetric flask.
• Place aliquots of 8ml into capped vials and freeze.
• Store frozen.

• Place exactly 9.2ml TEA (97%) mark to mark using a 10ml graduated pipette to 90.8 ml distilled water.
• Store for later use.

To construct above stock solution follow instructions below:

• Weigh 0.2500 g beta-sitosterol in a 150ml beaker.
• Add precisely 6.25 ml Tween 80 (use pipette to deliver).
• Stir and heat at low temp on a hot plate until dry chemical is dissolved.
• Turn off heat (keep stirring) and add 30ml distilled water.
• Remove from heat and place in a volumetric flask and bring to volume.
• Place 6ml of solution into small screw top autoclaveable tubes.
• Autoclave for 15min @ 122C, 15 psi, exhaust slow.
• After removing from autoclave, immediately vortex to combine the two layers.
• Cool to room temperature and tighten lids.
• Store in refrigerator.
• If precipitate form in stock solutions, try to dissolve by heating in a hot water bath, *** if precipitate fails to dissolve, discard solutions.

To combine above media:

• Weigh out the components successively into a 400ml beaker.
• Add 85.0 ml distilled water.
• Stir and heat to 50-60C for 3-4 hours or until dissolved.
• Cool to room temperature.

To combine above media:

• Weigh out the components and place successively into a 250ml beaker.
• Add 20ml distilled water.
• While stirring add 0.617ml of 10%KOH solvent.
• Add 65ml distilled water.
• Dissolve, heat mildly if necessary.
• Cool.

Weigh and place into a small dry covered beaker.

Weigh and place in a small dry covered beaker with myo-inositol.

Weigh and place in a small dry covered beaker with choline dihydrogen citrate.

Weigh and place in a small dry covered beaker.

To compile above media:

• Weigh components successively into a 400ml beaker.
• Add 170ml distilled water.
• Stir and heat to 50-60C for 3-4 hours or until dissolved.
• Cool to room temperature.

To compound CbMM: (makes 500ml 2X solution)

• Make sure all solutions are the same temperature.
• Place a 1000ml beaker on a magnetic stirrer/heating plate (start the stirring action when the first solution is added).
• Quantitatively transfer both of the amino acid solutions and the nucleic acid solution into the flask (wash each transfer beaker with no more than 3ml water).
• Quantitatively add the mixed salt solution into the 1000ml beaker (use no more than 2ml rinse water)(or pippet exact amount).
• Pipette 6.25 ml of each of the 2 vitamin solutions.
• Add successively the weighed ingredients (glutathione, choline and myo-inositol, cytochrome c, glucose or K-acetate) allow each to dissolve prior to adding the next dry component and use no more than 1-2ml of rinse water for each component.
• Add 10ml beta-sitosterol.
• Warm the medium slightly (30C) for about 1 hour to facilitate complete solution of all components.
• Adjust pH to 5.9 +- 1 with 10%KOH.
• Transfer to a 500ml volumetric flask and bring to volume with distilled water.
• Filter sterilize using 0.22micron pore size filter setup.
• Store in refrigerator at 2C in dark.

Standard Rules to follow concerning volumes

• The standard control solution for CbMM setups is water.
• The replicate number of controls should exceed the replicate number for each experimental extract.
• Standard experimental volume of CbMM is 2ml.
• Standard experimental volume of other extracts is 0.50ml and 0.20ml.
• Total experimental volume for CbMM experiments is 2.50ml.
• ID# for tube will contain extract volume, extract identification code or extract number, and replicate number.

• All bottles to be used for culture must be washed at least 2 times without soap in an automatic dishwasher.
• All bottle containers will be enclosed in aluminum foil prior to being autoclaved.
• All culture tubes must be autoclaved two times or 60 min.
• Aluminum foil will not be removed until the bottles have been placed in a laminar flow hood.
• Drying of bottles will take place either in the autoclave or in the laminar flow hood.
• When working with materials in the laminar flow hood, sterile technique should be followed at all times.
• Prior to use, the outsides of the bottles are sprayed with Lysol-IC.
• When caps are removed they are placed in sterile covered petrie dishes to prevent contamination.
• Once the lids are removed, at no time should anything but the sterile end of a pipet be placed above the open end of the bottle.
• experimental volume of CbMM is added to each bottle
• The experimental volume of extract is added to each bottle.
• Each bottle should now be shaken slightly to mix the contents together.
• Each culture should now be inoculated with nematodes.
• All culture bottles should be capped and closed.
• Culture bottle caps should be wrapped with parafilm in a clockwise direction.
• All culture tubes should now be marked with identification numbers.
• Tape should be placed around the caps to act as a safety seal.
• The number of nematodes in each experimental tube should now be counted and recorded.
• Record nematode number in each tube every 3-4 days.
• When experiment is finished record the pH of every tube.

RULES:

• Any movement of the nematode not consistent with browning motion or associated with movements of the fluid in the culture denotes a live nematode.
• Non-moving nematodes are presumed dead.
• Nematodes retained inside cadavers will not be counted until they have left the cadaver.
• No eggs will be counted as nematodes.
• All live nematodes in the culture are to be counted.
• Experimental cultures should be counted immediately after inoculation as time permits.
• Day zero is the inoculation day of the culture.
• If possible, cultures should be counted within 24 hours of inoculation to assure survivability of some nematodes in each experimental tube.
• Experimental cultures will continue to be counted every 3-4 days.
• Any contamination of the culture by bacteria or fungal growth will be noted.
• Any experimental cultures showing a zero inoculation number will continue to be counted during the run of the experiment to assure the lack of inoculation.
• No tube shall be re-inoculated after initial setup even if the inoculation number is determined to be zero.