CeHR MediumC. elegans Habitation and Reproduction (CeHR) Medium is a defined, bacterial-free medium that was developed at the USACEHR for culturing worms. It can be used in toxicological studies without the concern of bacterial metabolism of test chemicals confounding the experiment. Preparation of CeHR MediumI. Preparation of Stock Solutions:A. 10 X Salt Solution (prepared in 1 L) 1. To 800 mL of ultra-pure water add the following amounts in "Stock solution":
Adjust the volume of this stock solution to 1 liter with ultra-pure water. Sterilize by filtration and store at +4°C. B. 100X Vitamins and Growth Factors (prepared in 100 mL) 1. To 60 mL of ultra-pure water add the following amounts in "Stock solution":
2. To 5 mL of 1N KOH add the following amounts in "Stock solution":
Add directly to stock solution (from step 1). Dissolve the following in 1 mL ethanol and add to stock solution.
5. Bring stock solution to 100 mL with ultra-pure water. 6. Sterilize by filtration. Prepare 10 mL aliquots in sterile 15 mL tubes and store at -20°C. C. 50X Nucleic Acid Mix (use sodium salts - prepared in 100 mL) 1. To 60 mL of ultra-pure water add the following amounts in "Stock solution":
*NOTE: Thymine is difficult to get into solution. Gentle heating and stirring will help. 2. Bring to 100 mL. with ultra-pure water. 3. Sterilize solution by autoclaving (+121°C, 30 minutes). Store at +4°C. D. Other components, each prepared as a separate stock solution using ultra-pure water except as noted:
*NOTE: These reagents are light sensitive. Wrap in foil or store in amber bottles. 1. Each of these stock solutions is sterilized separately by filtration with the exception of the cholesterol, which is assumed to be sterile because of the ethanol. 2. All are stored at +4°C. II. Assembly of CeHR Medium (for 1 liter equivalent of medium)Measure 500 mL Milli-Q water. Prewet the filter on a 1 L sterile vacuum filtration unit with a small amount of the 500 mL of water. Add the following stocks one at a time to the filter unit with the vacuum on low: a. 10 mL Choline b. 10 mL 100 X Vitamins and Growth Factors c. 10 mL i-Inositol d. 10 mL Hemin chloride e. 10 mL Cytochrome C 4. Add about 250 mL of the premeasured water to the unit, then add the following: a. 20 mL 50X Nucleic acid mix b. 100 mL 10X Salt solution c. 20 mL Lactalbumin hydrolysate d. 20 mL Essential Amino Acid Mix (GIBCO #11130-051) e. 10 mL Non-essential Amino Acid Mix (GIBCO #11140-050) f. 20 mL KH2PO4 g. 50 mL d-Glucose h. 10 mL HEPES 5. Add the remaining water to the filter unit. 6. Once the water has all passed through the unit, disconnect it from the vacuum line and remove the filter. 7. Add 1 mL cholesterol stock to the mixture under sterile conditions. 8. Cap and store at +4°C. NOTE: Immediately before use, add 20% (v/v) ultra-pasteurized (HT) fat-free, organic milk. Several brands of milk have been tried. All appear to work. Organic milk is presumed to lack nematicides and other agricultural chemicals. III. Media pointersA. Always swirl media before using to start cultures. Several components precipitate out and settle to the bottom. Remember, you can't filter the media once it has been assembled and once milk has been added. If you do, you will be filtering out necessary components. B. Once the medium (w/o milk) has been assembled, the following steps should be performed to make sure it is up to par: 1. First, check the osmolality. Record the OS (should be 230 - 245). 2. Next, check the pH. Put 8mL into a 15mL conical vial and test the pH. Then add 2mL of milk to it, mix, and check the pH again. pH without milk should be 5.8 - 6.2. pH with milk should be 6.0 - 6.5. 3. You'll probably also want to streak a few microliters onto a small BHI +.5% Glucose plate, and incubate that at +37°C overnight. C. We use ultra-pasteurized, fat-free organic milk that is kept frozen (-80°C) in aliquots of ~220 mL (sufficient volume for 1 L of media). It should be sterile when opened. The half gallon container is opened using sterile technique, then the milk is streaked on BHI agar plates supplemented with glucose to check for sterility prior to use in CeHR medium. The milk is transferred to sterile plastic containers for storage at -80°C. D. Occasionally, a batch of ultra-pasteurized, fat-free milk has shown contamination when opened. Autoclaving the ultra-pasteurized, fat-free milk appears to negatively affect a necessary factor, resulting in either lengthened generation intervals or failure to reproduce. Similarly, attempts to autoclave and use non-sterile milk products such as pasteurized skim milk or reconstituted skim milk powder have not been as successful. The milk cannot be sterilized by filtration. Use of milk from which the milk fat has not been removed makes microscopic observation of the worms difficult due to the fat globules.
Last Modified Date: 02 Jul 2010
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