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C. elegans Habitation and Reproduction medium (CeHR-3)

United States Army Center for Environmental Health Research, Clegg lab

I. Preparation of the stock solutions 

Salt Solution, 1L

Dissolve the following components in 1L of water, and store at 4°C protected from light.

Temp Chemical Amount Source
rt MgCl2·6H2O 4.1 g Sigma M2393
rt Sodium Citrate 2.9 g Sigma S4641
rt Potassium Citrate·H2O 4.9 g Sigma P1722
rt CuCl2·2H2O 0.07 g Fisher C455-500
rt MnCl2·4H2O 0.2 g Fisher M87-100
rt ZnCl2 0.1 g Sigma Z0152
rt Fe(NH4)2(SO4)2·6H2O 0.6 g Sigma F1018
rt CaCl2·2H2O 0.2 g Fisher C70-500

Vitamins and Growth Factors

First dissolve the following components in 5 ml of 1N KOH

Temp Chemical Amount Source
4°C p-Aminobenzoic Acid 0.075 g Sigma A9878
4°C Biotin 0.0375 g Sigma B4639
4°C Cyanocobalamine (B-12) 0.0375 g Sigma V2876
RT Folinate (Ca) (Folinic Acid) 0.0375 g Sigma F7878
RT Niacin / Nicotin 0.075 g Sigma N0761
-20°C Pyridoxal 5'-phosphate 0.0375 Sigma P3657

 

Then bring the volume up to 100 ml with water and add the following components

Temp Chemical Amount Source
4°C N-Acetylglucosamine 0.15 g Calbiochem 1079
RT DL-Alanine 0.15 g Calbiochem 125005
RT Niacinamide / Nicotinamide 0.075 g Sigma N3376
4°C Pantetheine 0.0375 Sigma P2125
4°C Pantothenate (Ca) 0.075 Sigma P6292
4°C Pteroylglutamic Acid (Folic Acid) 0.075 Sigma 47620
-20°C Pyridoxamine·2HCl 0.0375 Sigma P9158
RT Pyridoxine·HCl 0.075 Sigma P6280
-20°C Riboflavin 5-PO4(Na) 0.075 Sigma R7774
RT Thiamine·HCl 0.075 Sigma T1270

 

Finally, dissolve Thiocitic Acid in 1 ml ethanol and add to the stock solution.

Temp Chemical Amount Source
RT DL-6,8-Thiocitic Acid / αLipoic Acid 0.0375 Sigma T1395

 

Store frozen or protected from light at 4°C.

Nucleic Acid substituents

Dissolve the following components in 100 ml of water, and store at 4°C protected from light or frozen.

Temp Chemical Amount Source
-20°C Adenosine 2'- & 3'-PO4 or 5'-PO4 1.74 g Sigma A1752
4°C Cytidine 5'-PO4 1.84 g Sigma C1006
4°C Guanosine 5'-PO4 2.04 g Sigma G8377
4°C Uridine 5'-PO4 1.84 g Sigma U6375

 

Dissolve Thymine in 5ml 1N NaOH, then increase volume to 100 ml with water and store at 4°C protected from light or frozen.

Temp Chemical Amount Source
-20°C Thymine 0.63 g Sigma T0376

Additional stock solutions

All solutions are stored at 4°C protected from light.

Temp Chemical Amount Source
RT KH2PO4 61.3 g/L in water Sigma P5379
RT Choline di-acidic citrate 5.9 g/100 ml in water Sigma C2004
RT i-Inositol 4.32 g/100 ml in water Sigma I5125
RT d-Glucose 263 g/L in water Sigma G7520
-20°C Cytochrome c 0.1 g/20 ml in water Sigma C7752
RT Hemin Chloride 0.1 g/100 ml in 0.1 N NaOH Calbiochem 3741
RT Hepes, Na salt 1 M stock in water Sigma H3784
RT Cholesterol 5 mg/ml in EtOH
RT Lactalbumin hydrolysate 17 g/100 ml in water Sigma L9010

Additional solutions to purchase

Essential amino-acid mix: Invitrogen/Gibco 11130-051

Non-Essential amino-acid mix: Invitrogen/Gibco 11140-050

 

II Assembly of the medium

For 1 L of medium, filter the following volumes of stock solutions and water through a 0.22 µm filter in the order described

Amount Solution
10 ml Choline
10 ml Vitamin mix
10 ml i-Inositol
10 ml Hemin Chloride
10 ml Cytochrome c
250 ml Water
20 ml Nucleic acid mix
20 ml Thymine
100 ml Salt solution
20 ml Lactalbumin Hydrolysate
20 ml Essential amino-acids
10 ml Non-essential amino-acids
20 ml KH2PO4
50 ml d-Glucose
10 ml Hepes
250 ml Water

 

Add 1 ml cholesterol and store at 4°C protected from light.

Before use add 200 ml ultra-pasteurized skim milk. The Clegg lab recommends a product stored at 4°C, which I could not find in my grocery store. I've used parmalat with no problems.