Modified C. elegans Habitation and Reproduction medium (mCeHR medium). 

CeHR medium was originally formulated by Dr. Eric Clegg (http://usacehr.detrick.army.mil/CleggLab.html) at the United States Army Center for Environmental Health Research to grow worms in an axenic liquid media.

Reference: Clegg, E. D., LaPenotiere, H. F., French, D. Y. & Szilagyi, M. Use of CeHR Axenic Medium for Exposure and Gene Expression Studies. East Coast Worm Meeting, Abstract 91 (2002).

We are thankful to Eric for his invaluable help and advice through all the modifications we have since made to optimize CeHR medium to suit our research goals. We modified CeHR medium (mCeHR) because of our interests in defining the individual micronutrient components in order to identify the cellular and molecular components of metal homeostasis using C. elegans as a model multicellular eukaryote.

Preparation of Solutions:

Water used is generated from a Milli-Q system.

a) To make 5 N NaOH, measure 50.0 g NaOH into a 250 ml volumetric flask and bring to volume with water.  Store at room temperature.
b) To make 0.1 M NaCl, measure 5.8 g NaCl into a 1 L volumetric flask and bring to volume with water.  Sterilize by autoclaving at 121^oC for 15 min.
c) To make M9 buffer, measure 6.0g Na₂HPO₄ (42 mM), 3.0g KH₂PO₄ (22 mM), 5.0g NaCl (86 mM) and 0.25g MgSO₄.7H₂O (1mM) into a 1 L volumetric flask and bring to volume with water. Sterilize by autoclaving at 121^oC for 15 min.

Note: store solutions b and c at 4º

d) Hemin chloride stock solution (Mol. Wt: 651.96.):

• Solutions of concentration up to 2mM can be made in 0.1 N NaOH, but higher concentrations should be made in 0.3M NH₄OH. (Do not make it to the required volume as the solution needs to adjusted to a pH in the range of 7.4-8.0.)
• Adjust the pH with 6N HCl stirring constantly. A very small volume of HCl is required to adjust the pH.
• Make up the volume with 0.3M NH₄OH, pH 8.0.
• If the pH goes below 6.0 (acidic), hemin will precipitate out.
• The same procedure is followed for making up any of the metal substituted porphyrins.  Meso- is harder to go into solution then the proto- porphyrin.

Procedure:

For Worm Egg Prep:

1.      All work must be carried out using sterile technique in a laminar flow hood.
2.      Pipette nematode suspension into sterile conical tubes and centrifuge at 800 xg for 5 min at 4^oC.
3.      Aspirate supernatant using a Pasteur pipette and resuspend nematode pellet in 10 ml of 0.1 M NaCl by gentle pipeting.  Incubate nematodes on ice for 5 min.
4.      Aspirate supernatant using a Pasteur pipette, including worms that have not settled to the bottom of the tube.
5.      Add 0.1 M NaCl up to a volume such that the worm suspension is not very dense. You should be able to see individual worms floating/swimming around. The volume of NaCl should be in multiples of 3. For example, 1.5 ml, 3 ml, 6ml, 9 ml, etc..
6.      Mix one part 5 N NaOH with 2 parts 5 % Chlorox Bleach (concentrated bleach should be diluted to 5 % in water).  USE IMMEDIATELY!  The volume of bleach required is determined by the volume of 0.1 M NaCl used in the previous step.
7.      Add NaOH/bleach mix to nematode population (The volume of bleach/NaOH added should be half the volume of worms). 
For example: If you have the worms in 6 ml of 0.1 M NaCl, then you should add 2 ml of 5 % bleach to 1 ml of NaOH and add the total 3 ml to 6 ml of worms.
8.      Vortex at max setting (#10 on a vortexGenie).
9.      Observe the worms under a microscope with occasional vortexing at high speeds.
10.  The bleaching is complete when you can see the worms pop-open at the vulva or the mouth to release the eggs. It should take less than 10 min for the worms to open-up.
11.  Let the worms stay in bleach for an additional 30-40 sec for the worms to dissolve.
12.   Immediately centrifuge nematodes at 800 xg for 45 sec at 4^oC in a tabletop clinical centrifuge. 
13.  Aspirate supernatant using a Pasteur pipette.
14.  Add sterile water to egg pellet to a volume of 5-10 ml depending on pellet size and vortex tube at setting 10 for 5 sec. Centrifuge nematodes at 800 g for 45 sec at 4^oC. Aspirate supernatant using a Pasteur pipette.
15.  Repeat step 14.
16.  Add 10 (30) ml M9 salt solution to eggs and transfer to sterile T-25 (T-75) cm² cell culture flask [Fisher #12-565-31/57]. 
17.  Incubate flask at 20^oC ± 1^oC on a shaker platform at 70 rpm.  The eggs will hatch in the M9 buffer and remain as L1 larvaes till growth media is added (we like to use the L1s within a week).

Preparation of CeHR-3 Medium

I. Preparation of Stock Solutions:  A. Salt Solution (Mineral Mix) prepared in 1 L

To 800 ml of ultra-pure water add the amounts under Stock solution:

Adjust the volume of this stock solution to 1 liter with ultra-pure water and store at 4^oC.

B. Vitamins and Growth Factor mix prepared in 100 ml

To 60 ml of ultra-pure water add the following to make the stock solution:

 

Chemical	Final conc. in CeHR (mg/L)	Final Conc. in CeHR (uM)	Stock solution (grams)	Source	Cat #
N-Acetylglucosamine	15	67.81	0.15	Calbiochem	1079
DL-Alanine	15	168.35	0.15	Calbiochem	125005
p-Aminobenzoic Acid*	7.5	54.69	0.075	Sigma	A-9878
Biotin*	3.75	15.35	0.0375	Sigma	B-4639
Cyanocobalamine (B-12)*	3.75	2.77	0.0375	Sigma	V--2876
Folinate (Ca) *	3.75	7.33	0.0375	Sigma	F-7878
Niacin*	7.5	60.92	0.075	Sigma	N-0761
Niacinamide	7.5	61.41	0.075	Sigma	N-3376
Pantetheine	3.75	6.76	0.0375	Sigma	P-2125
Pantothenate (Ca)	7.5	31.47	0.075	Sigma	P-6292
Pteroylglutamic Acid (Folic Acid)	7.5	16.99	0.075	ACRCS	21663-0100
Pyridoxal 5'-phosphate*	3.75	15.18	0.0375	Sigma	P-3657
Pyridoxamine.2HCl	3.75	15.55	0.0375	Sigma	P-9158
Pyridoxine.HCl	7.5	36.48	0.075	Sigma	P-6280
Riboflavin 5-PO4(Na)	7.5	15.68	0.075	Sigma	R-7774
Thiamine.HCl	7.5	22.24	0.075	Sigma	T-1270
DL-6,8-Thioctic Acid**	3.75	18.18	0.0375	Sigma	T-1395

 

 

 

 

 

 

 

 

 

 

 

 

* Dissolve in ~5 ml 1N KOH and add to stock solution
** Dissolve in ~1 ml ethanol, then add to stock solution
Bring stock solution to 100 ml with water and store in bottle covered with aluminum foil at 4^oC or freeze aliquots at -20^oC.

C. Nucleic acid mix (use sodium salts) prepared in 100 ml

To 60 ml of ultra-pure water add the following:

Bring to 100 ml. with ultra-pure water and store at 4^oC or freeze aliquots at -20^oC.

D. Other components are prepared as separate stock solutions using ultra-pure water except as noted.
 

All solutions are stored at 4^oC.
Stocks do not have to be filtered because they will be filtered together to make the media.

II.  Assembly of CeHR Medium

NOTE: make small volumes of stocks so they are fresh. Make media the day before use and only make what is needed. Make sure to record the pH. The pH without milk is 6.0-6.5.  We assemble all contents in a laminar flow hood (tissue culture hood). 

For 1 L medium:

Using sterile technique and a 1L (0.2 mm) vacuum filter unit, filter the following volumes of stock solutions and water in the order described. (Amounts in parentheses indicate volumes for 750 ml.)

10 ml Choline (7.5 ml)
10 ml Vitamin mix (7.5 ml)
10 ml i-Inositol (7.5 ml)
10 ml Hemin chloride
250 ml Water
20 ml Nucleic acid mix (15 ml)
100 ml Salt solution (75 ml)
20 ml Lactalbumin hydrolysate (GIBCO #18080-036)*** (15 ml)
20 ml Essential Amino Acid Mix (GIBCO #11130-051) (15 ml)
10 ml Non-essential Amino Acid Mix (GIBCO #11140-050) (7.5 ml)
20 ml KH₂PO₄ (15 ml)
50 ml d-Glucose (37.5 ml)
10 ml HEPES (7.5 ml)
250 ml Water

Total volume to this point is 800 ml.

Remove filter unit from vacuum and add 1 ml cholesterol.  Store at 4 ^oC.

*** If unavailable, substitute 8.5 g of Sigma L-9010 in 50 ml dH₂0 for a 50X stock.

Add 200 ml sterile high temperature ultra-pasteurized skim milk to the above mixture at time of use.  UHT skim (fat free) milk – 20% (v/v)

Notes:

High temperature ultra-pasteurized (UHT) skim milk can be obtained in a grocery store (Eg: Horizon’s Organic UHT skim milk). We use a product that is kept refrigerated (the room temperature-stored product does not work well). It should be sterile when opened. The half-gallon container is opened using sterile technique, then the milk is streaked on Brain Heart Infusion (BHI) [DIFCO #237500] agar plates to check for sterility prior to use in CeHR medium. The milk is transferred to 50 ml sterile tubes and stored at -80^oC.

NOTE: Make sure to plate milk first on BHI to see if microbes are present.

 

Last updated by I.H. Feb 2005 (Email: hamza@umd.edu)

Hamza Lab Website